Photolabile compounds

ABSTRACT

The present invention describes Photolabile Compounds methods for use of the compounds. The Photolabile Compounds have a photoreleasable ligand, which can be biologically active, and which is photoreleased from the compound upon exposure to light. In some embodiments, the Photolabile Compounds comprise a light antenna, such as a labeling molecule or an active derivative thereof. In one embodiment, the light is visible light, which is not detrimental to the viability of biological samples, such as cells and tissues, in which the released organic molecule is bioactive and can have a therapeutic effect. In another embodiment, the photoreleasable ligand can be a labeling molecule, such as a fluorescent molecule.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of International PatentApplication No. PCT/US2010/40220, filed Jun. 28, 2010, which claimspriority to U.S. Provisional Patent Application Ser. No. 61/220,922,filed Jun. 26, 2009, the entire disclosure of each is incorporated byreference herein. U.S. Pat. Pub. No. 2008-0176940 is also incorporatedherein by reference in its entirety.

The work herein was supported in whole, or in part, by NationalInstitute of Health Grant Nos. EY011787 and EY013237. Thus, the UnitedStates Government has certain rights to the invention. This inventionwas made with government support under NYSTAR Contract No. C000082awarded by NYSTAR. The government has certain rights in the invention.

This patent disclosure contains material which is subject to copyrightprotection. The copyright owner has no objection to the facsimilereproduction by anyone of the patent document or the patent disclosure,as it appears in the U.S. Patent and Trademark Office patent file orrecords, but otherwise reserves any and all copyright rights.

FIELD OF THE INVENTION

The present invention relates generally to novel Photolabile Compoundsand methods for uncaging an organic molecule, such as a bioactivemolecule, which can have a variety of uses both in vitro and in vivo.

BACKGROUND OF THE INVENTION

Photolabile protecting groups, which are also called caging groups, areclasses of protecting groups that are particularly useful in thebiological sciences. Because light can be controlled with precision bothspatially and temporally, cleaving a protecting group from a bioactivemolecule allows release, or uncaging, of the molecule. Protecting groupstypically mask or conceal charged (for example, carboxylate orphosphate) or polar (for example, amine, hydroxyl, or sulfhydryl) groupson the compounds. Frequently such functionalities increase thehydrophobicity and membrane permeability of the protected molecules.Prior to photolysis, the Photolabile Compounds are typically chemicallyor biologically inactive because at least one of the compounds' mainfunctionalities is blocked. The activity of the molecule can betriggered by a pulse of light, thereby releasing the molecule from thephotoreleasable compound. Thus, photolabile protecting groups can beremoved from a protected compound by irradiation, for example, tocontrol the release of the compound when and where desired, either invivo or in vitro.

Commercially available Photolabile Compounds typically requireultraviolet (UV) light to remove the compounds from the cage. However,UV light can cause damage to organs, tissues and cells, thus making UVlight detrimental for in vivo use. Thus, there is a need in the art toutilize new Photolabile Compounds having ligands that can be releasedusing light other than UV light, particularly for in vivo applications.The present invention provides novel Photolabile Compounds and methodsfor using the compounds, which provide advantages over currentlyavailable compounds that are photolabile using only UV light.

SUMMARY OF THE INVENTION

It is an aspect of the present invention to provide novel PhotolabileCompounds that protect an organic molecule, such as a bioactivemolecule. Upon exposure to light, the organic molecule is released, andis useful in the methods described herein.

In one aspect, the present invention provides compounds of Formula I:

wherein M is Ru;

each L¹ is independently an organic molecule having:

(a) a 5-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with M;

(b) a 6-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with M;

(c) an 8-10-membered bicyclic ring, one of the bicyclic rings beingaromatic and having a nitrogen atom member that forms a bond with M;

(d) an —NH₂ group whose nitrogen atom forms a bond with M;

(e) a —COOH group, one of whose oxygen atoms forms a bond with M; or

(f) a —CN group whose nitrogen atom forms a bond with M;

L² is (R⁹)₃P, (R⁹O)₃P, or L¹ and m is 2; or L² is —CN and m is 1; or L²is a labeling molecule or an active derivative thereof connected to Mthrough the phosphorous atom of (R⁹)₂P or (R⁹O)₂P and m is 2; or L² is alabeling molecule or an active derivative thereof connected to M throughthe nitrogen atom of: NHR⁹, N(R⁹)₂, pyridyl, C(R⁹)═NH, C(R⁹)═NR⁹ cyclicaliphatic amine group or nitrile and m is 2;

wherein each R⁹ is independently —C₁-C₁₈ alkyl, —C₃-C₈ cycloalkyl, orphenyl;

R¹-R⁸ are independently —H, —C₁-C₁₈ alkyl, —NH₂, —COOH, —(C₁-C₁₈alkyl)-O—(C₁-C₁₈ alkyl), or —OC(O)(C₁-C₁₈ alkyl); and

X is Cl⁻, F⁻, Br⁻, I⁻, PF₆ ⁻, CF₃SO₃ ⁻, (C₁-C₁₈ alkyl)-CO₂ ⁻, or (C₁-C₁₈alkyl)-SO₃ ⁻.

In one embodiment of the compounds of Formula I,

M is Ru;

each L¹ is independently an organic molecule having:

(a) a 5-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with M;

(b) a 6-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with M;

(c) an 8-10-membered bicyclic ring, one of the bicyclic rings beingaromatic and having a nitrogen atom member that forms a bond with M;

(d) an —NH₂ group whose nitrogen atom forms a bond with M; or

(e) a —COOH group, one of whose oxygen atoms forms a bond with M;

L² is (R⁹)₃P, (R⁹O)₃P, or L¹ and m is 2; or L² is —CN and m is 1; or L²is a labeling molecule or an active derivative thereof connected to Mthrough the phosphorous atom of (R⁹)₂P or (R⁹O)₂P and m is 2; or L² is alabeling molecule or an active derivative thereof connected to M throughthe nitrogen atom of: NHR⁹, N(R⁹)₂, pyridyl, C(R⁹)═NH, C(R⁹)═NR⁹ cyclicaliphatic amine group or nitrile and m is 2;

wherein each R⁹ is independently —C₁-C₁₈ alkyl, —C₃-C₈ cycloalkyl, orphenyl;

R¹-R⁸ are independently —H, —C₁-C₁₈ alkyl, —NH₂, —COOH, —(C₁-C₁₈alkyl)-O—(C₁-C₁₈ alkyl), or —OC(O)(C₁-C₁₈ alkyl); and

X is Cl⁻, F⁻, Br⁻, I⁻, PF₆ ⁻, CF₃SO₃ ⁻, (C₁-C₁₈ alkyl)-CO₂ ⁻, or (C₁-C₁₈alkyl)-SO₃ ⁻.

In one embodiment of the compounds of Formula I,

M is Ru;

each L¹ is independently an organic molecule having:

(a) a 5-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with M;

(b) a 6-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with M;

(c) an 8-10-membered bicyclic ring, one of the bicyclic rings beingaromatic and having a nitrogen atom member that forms a bond with M;

(d) an —NH₂ group whose nitrogen atom forms a bond with M;

(e) a —COOH group, one of whose oxygen atoms forms a bond with M; or

(f) a —CN group whose nitrogen atom forms a bond with M;

L² is (R⁹)₃P, (R⁹O)₃P, or L¹ and m is 2; or L² is —CN and m is 1;

R¹-R⁸ are independently —H, —C₁-C₁₈ alkyl, —NH₂, —COOH, —(C₁-C₁₈alkyl)-O—(C₁-C₁₈ alkyl), or —OC(O)(C₁-C₁₈ alkyl); and

X is Cl⁻, F⁻, Br⁻, I⁻, PF₆ ⁻, CF₃SO₃ ⁻, (C₁-C₁₈ alkyl)-CO₂ ⁻, or (C₁-C₁₈alkyl)-SO₃ ⁻.

In one embodiment of the compounds of Formula I,

M is Ru;

each L¹ is independently an organic molecule having:

(a) a 5-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with M;

(b) a 6-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with M;

(c) an 8-10-membered bicyclic ring, one of the bicyclic rings beingaromatic and having a nitrogen atom member that forms a bond with M;

(d) an —NH₂ group whose nitrogen atom forms a bond with M; or

(e) a —COOH group, one of whose oxygen atoms forms a bond with M;

L² is (R⁹)₃P, (R⁹O)₃P, or L¹ and m is 2; or L² is —CN and m is 1;

R¹-R⁸ are independently —H, —C₁-C₁₈ alkyl, —NH₂, —COOH, —(C₁-C₁₈alkyl)-O—(C₁-C₁₈ alkyl), or —OC(O)(C₁-C₁₈ alkyl); and

X is Cl⁻, F⁻, Br⁻, I⁻, PF₆ ⁻, CF₃SO₃ ⁻, (C₁-C₁₈ alkyl)-CO₂ ⁻, or (C₁-C₁₈alkyl)-SO₃ ⁻.

In some embodiments of the compounds of Formula I, when L² is (R⁹)₃P,each R⁹ is different from each other.

In some embodiments of the compounds of Formula I, when L² is (R⁹)₃P,two R⁹ are the same and different from the remaining R⁹.

In some embodiments of the compounds of Formula I, when L² is (Ph)₃P,each phenyl is not substituted with methyl.

In another aspect, the present invention provides compounds of FormulaII:

wherein:

each L¹ is independently an organic molecule having:

(a) a tetrazolyl group, one of its nitrogen atoms forming a bond withRu;

(b) nicotine or caffeine, whose pyridyl nitrogen atom forms a bond withRu;

(c) an 8-10-membered bicyclic ring, one of the bicyclic rings beingaromatic and having a nitrogen atom member that forms a bond with Ru;

(d) an —NH₂ group whose nitrogen atom forms a bond with Ru;

(e) a —COOH group, one of whose oxygen atoms' forms a bond with Ru; or

(f) a —CN group whose nitrogen atom forms a bond with Ru;

L² is (R⁹)₃P, (R⁹O)₃P, or Land m is 2; or L² is —CN and m is 1; or L² isa labeling molecule or an active derivative thereof connected to Ruthrough the phosphorous atom of (R⁹)₂P or (R⁹O)₂P and m is 2; or L² is alabeling molecule or an active derivative thereof connected to Ruthrough the nitrogen atom of: NHR⁹, N(R⁹)₂, pyridyl, C(R⁹)═NH, C(R⁹)═NR⁹cyclic aliphatic amine group or nitrile and m is 2;

wherein each R⁹ is independently —C₁-C₁₈ alkyl, —C₃-C₈ cycloalkyl, orphenyl;

R¹-R⁸ are independently —H, —C₁-C₁₈ alkyl, —NH₂, —COOH, —(C₁-C₁₈ alkyl),or —OC(O)(C₁-C₁₈ alkyl); and

X is Cl⁻, F⁻, Br⁻, I⁻, PF₆ ⁻, CF₃SO₃ ⁻, (C₁-C₁₈ alkyl)-CO₂ ⁻, or (C₁-C₁₈alkyl)-SO₃ ⁻.

In one embodiment of the compounds of Formula II,

each L¹ is independently an organic molecule having:

(a) a tetrazolyl group, one of its nitrogen atoms forming a bond withRu;

(b) nicotine or caffeine, whose pyridyl nitrogen atom forms a bond withRu;

(c) an 8-10-membered bicyclic ring, one of the bicyclic rings beingaromatic and having a nitrogen atom member that forms a bond with Ru;

(d) an —NH₂ group whose nitrogen atom forms a bond with Ru; or

(e) a —COOH group, one of whose oxygen atoms forms a bond with Ru;

L² is (R⁹)₃P, (R⁹O)₃P, or Land m is 2; or L² is —CN and m is 1; or L² isa labeling molecule or an active derivative thereof connected to Ruthrough the phosphorous atom of (R⁹)₂P or (R⁹O)₂P and m is 2; or L² is alabeling molecule or an active derivative thereof connected to Ruthrough the nitrogen atom of: NHR⁹, N(R⁹)₂, pyridyl, C(R⁹)═NH, C(R⁹)═NR⁹cyclic aliphatic amine group or nitrile and m is 2;

wherein each R⁹ is independently —C₁-C₁₈ alkyl, —C₃-C₈ cycloalkyl, orphenyl;

R¹-R⁸ are independently —H, —C₁-C₁₈ alkyl, —NH₂, —COOH, —(C₁-C₁₈alkyl)-O—(C₁-C₁₈ alkyl), or —OC(O)(C₁-C₁₈ alkyl); and

X is Cl⁻, F⁻, Br⁻, I⁻, PF₆ ⁻, CF₃SO₃ ⁻, (C₁-C₁₈ alkyl)-CO₂ ⁻, or (C₁-C₁₈alkyl)-SO₃ ⁻.

In one embodiment of the compounds of Formula II,

each L¹ is independently an organic molecule having:

(a) a tetrazolyl group, one of its nitrogen atoms forming a bond withRu;

(b) nicotine or caffeine, whose pyridyl nitrogen atom forms a bond withRu;

(c) an 8-10-membered bicyclic ring, one of the bicyclic rings beingaromatic and having a nitrogen atom member that forms a bond with Ru;

(d) an —NH₂ group whose nitrogen atom forms a bond with Ru;

(e) a —COOH group, one of whose oxygen atoms forms a bond with Ru; or

(f) a —CN group whose nitrogen atom forms a bond with Ru;

L² is (R⁹)₃P, (R⁹O)₃P, or L¹ and m is 2; or L² is —CN and m is 1;

R¹-R⁸ are independently —H, —C₁-C₁₈ alkyl, —NH₂, —COOH, —(C₁-C₁₈alkyl)-O—(C₁-C₁₈ alkyl), or —OC(O)(C₁-C₁₈ alkyl); and

X is Cl⁻, F⁻, Br⁻, I⁻, PF₆ ⁻, CF₃SO₃ ⁻, (C₁-C₁₈ alkyl)-CO₂ ⁻, or (C₁-C₁₈alkyl)-SO₃ ⁻.

In one embodiment of the compounds of Formula II,

each L¹ is independently an organic molecule having:

(a) a tetrazolyl group, one of its nitrogen atoms forming a bond withRu;

(b) nicotine or caffeine, whose pyridyl nitrogen atom forms a bond withRu;

(c) an 8-10-membered bicyclic ring, one of the bicyclic rings beingaromatic and having a nitrogen atom member that forms a bond with Ru;

(d) an —NH₂ group whose nitrogen atom forms a bond with Ru; or

(e) a —COOH group, one of whose oxygen atoms forms a bond with Ru;

L² is (R⁹)₃P, (R⁹O)₃P, or L¹ and m is 2; or L² is —CN and m is 1;

R¹-R⁸ are independently —H, —C₁-C₁₈ alkyl, —NH₂, —COOH, —(C₁-C₁₈alkyl)-O—(C₁-C₁₈ alkyl), or —OC(O)(C₁-C₁₈ alkyl); and

X is Cl⁻, F⁻, Br⁻, I⁻, PF₆ ⁻, CF₃SO₃ ⁻, (C₁-C₁₈ alkyl)-CO₂ ⁻ or (C₁-C₁₈alkyl)-SO₃ ⁻.

In some embodiments of the compounds of Formula II, when L² is (R⁹)₃P,each R⁹ is different from each other.

In some embodiments of the compounds of Formula II, when L² is (R⁹)₃P,two R⁹ are the same and different from the remaining R⁹.

In some embodiments of the compounds of Formula II, when L² is (Ph)₃P,each phenyl is not substituted with methyl.

In another aspect, the present invention provides compounds of FormulaIII:

wherein:

L¹ is 4-aminopyridine (4-AP), whose pyridyl nitrogen atom forms a bondwith Ru;

L² is (R⁹)₃P, (R⁹O)₃P, or L¹ and m is 2; or L² is —CN and m is 1; or L²is a labeling molecule or an active derivative thereof connected to Ruthrough the phosphorous atom of (R⁹)₂P or (R⁹O)₂P and m is 2; or L² is alabeling molecule or an active derivative thereof connected to Ruthrough the nitrogen atom of: NHR⁹, N(R⁹)₂, pyridyl, C(R⁹)═NH, C(R⁹)═NR⁹cyclic aliphatic amine group or nitrile and m is 2;

wherein each R⁹ is independently —C₁-C₁₈ alkyl, —C₃-C₈ cycloalkyl, orphenyl;

R¹-R⁸ are independently —H, —C₁-C₁₈ alkyl, —NH₂, —COOH, —(C₁-C₁₈alkyl)-O—(C₁-C₁₈ alkyl), or —OC(O)(C₁-C₁₈ alkyl); and

X is a Cl⁻, F⁻, Br⁻, I⁻, PF₆ ⁻, CF₃SO₃ ⁻, (C₁-C₁₈ alkyl)-CO₂ ⁻, or(C₁-C₁₈ alkyl)-SO₃ ⁻.

In one embodiment of the compounds of Formula III,

L¹ is 4-aminopyridine (4-AP), whose pyridyl nitrogen atom forms a bondwith Ru;

L² is (R⁹)₃P, (R⁹O)₃P, or L¹ and m is 2; or L² is —CN and m is 1;

R¹-R⁸ are independently —H, —C₁-C₁₈ alkyl, —NH₂, —COOH, —(C₁-C₁₈alkyl)-O—(C₁-C₁₈ alkyl), or —OC(O)(C₁-C₁₈ alkyl); and

X is Cl⁻, F⁻, Br⁻, I⁻, PF₆ ⁻, CF₃SO₃ ⁻, (C₁-C₁₈ alkyl)-CO₂ ⁻, or (C₁-C₁₈alkyl)-SO₃ ⁻.

In some embodiments of the compounds of Formula III, when L² is (R⁹)₃P,each R⁹ is different from each other.

In some embodiments of the compounds of Formula III, when L² is (R⁹)₃P,two R⁹ are the same and different from the remaining R⁹.

In some embodiments of the compounds of Formula III, when L² is (Ph)₃P,each phenyl is not substituted with methyl.

In another aspect, the present invention provides compounds of FormulaIV:

wherein:M¹ is Li⁺, Na⁺, or K⁺;M² is Ru;and L¹ is independently an organic molecule having:

(a) a 5-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with M²;

(b) a 6-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with M²;

(c) an 8-10-membered bicyclic ring, one of the bicyclic rings beingaromatic and having a nitrogen atom member that forms a bond with M²;

(d) an —NH₂ group whose nitrogen atom forms a bond with M²;

(e) a —COOH group, one of whose oxygen atoms forms a bond with M²; or

(f) a —CN group whose nitrogen atom forms a bond with M².

In one embodiment of the compounds of Formula IV,

M¹ is Li⁺, Na⁺, or K⁺;

M² is Ru;

and L¹ is independently an organic molecule having:

(a) a 5-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with M²;

(b) a 6-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with M²;

(c) an 8-10-membered bicyclic ring, one of the bicyclic rings beingaromatic and having a nitrogen atom member that forms a bond with M²;

(d) an —NH₂ group whose nitrogen atom forms a bond with M²; or

(e) a —COOH group, one of whose oxygen atoms forms a bond with M².

In another aspect, the present invention provides compounds of FormulaV:

wherein:

each L¹ is independently an organic molecule having:

(a) a 5-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with Ru;

(b) a 6-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with Ru;

(c) an 8-10-membered bicyclic ring, one of the bicyclic rings beingaromatic and having a nitrogen atom member that forms a bond with Ru;

(d) an —NH₂ group whose nitrogen atom forms a bond with Ru;

(e) a —COOH group, one of whose oxygen atoms forms a bond with Ru;

(f) a —PR₂ group whose phosphorus atom forms a bond with Ru, wherein Ris independently —H, —C₁-C₁₈ alkyl, or aryl;

(g) an —SR group whose sulfur atom forms a bond with Ru, wherein R isindependently —H, —C₁-C₁₈ alkyl, or aryl; or

(h) a —CN group whose nitrogen atom forms a bond with Ru;

L² is (R⁹)₃P, (R⁹O)₃P, or L¹ and m is 2; or L² is —CN and m is 1; or L²is a labeling molecule or an active derivative thereof connected to Ruthrough the phosphorous atom of (R⁹)₂P or (R⁹O)₂P and m is 2; or L² is alabeling molecule or an active derivative thereof connected to Ruthrough the nitrogen atom of: NHR⁹, N(R⁹)₂, pyridyl, C(R⁹)═NH, C(R⁹)═NR⁹cyclic aliphatic amine group or nitrile and m is 2;

wherein each R⁹ is independently —C₁-C₁₈ alkyl, —C₃-C₈ cycloalkyl, orphenyl;

wherein when L² is P(phenyl)₃ or a labeling molecule or an activederivative thereof connected to Ru through the phosphorous atom ofP(phenyl)₂, each phenyl is independently substituted with —C₁-C₁₈ alkyl,—(C₁-C₁₈ alkyl)-OH, aryl, —(C₁-C₁₈ alkyl)-oxy, —(C₁-C₁₈ alkyl)-amino,—(C₁-C₁₈ alkyl)-thio, —CO₂Y, —C(═O)Y, —C(═O)NY₂, —NH₂, —NO₂, —OH, or—SH, and

R¹ to R⁴ are independently —H, —C₁-C₁₈ alkyl; —NH₂, —(C₁-C₁₈alkyl)-O—(C₁-C₁₈ alkyl), —OC(O)(C₁-C₁₈ alkyl), —(C₁-C₁₈ alkyl)-OH, aryl,—(C₁-C₁₈ alkyl)-oxy, —(C₁-C₁₈ alkyl)-amino, —(C₁-C₁₈ alkyl)-thio, —CO₂Y,—C(═O)Y, —C(═O)NY₂, —NO₂, or —SH, or R¹ and R² and/or R³ and R⁴ cancombine to form a carbocyclic ring substituted by one or more oxogroups;

wherein when L² is not P(phenyl)₃, R¹ to R⁴ are independently —H,—(C₁-C₁₈ alkyl)-OH, aryl, —(C₁-C₁₈ alkyl)-oxy, —(C₁-C₁₈ alkyl)-amino,—(C₁-C₁₈ alkyl)-thio, —CO₂Y, —C(═O)Y, —C(═O)NY₂, —NO₂, —OH, or —SH, orR¹ and R² and/or R³ and R⁴ can combine to form a carbocyclic ringsubstituted by one or more oxo groups, wherein at least one of R¹ to R⁴is not H;

X is Cl⁻, F⁻, Br⁻, I⁻, PF₆ ⁻, CF₃SO₃ ⁻, (C₁-C₁₈ alkyl)-CO₂ ⁻, or (C₁-C₁₈alkyl)-SO₃ ⁻; and

Y is selected from the group consisting of —H, —C₁-C₁₈ alkyl, aryl,—(C₁-C₁₈ alkyl)-aryl, —C₃-C₈ cycloalkyl, heteroaryl, and heterocyclyl.

In one embodiment of the compounds of Formula V:

each L¹ is independently an organic molecule having:

(a) a 5-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with Ru;

(b) a 6-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with Ru;

(c) an 8-10-membered bicyclic ring, one of the bicyclic rings beingaromatic and having a nitrogen atom member that forms a bond with Ru;

(d) an —NH₂ group whose nitrogen atom forms a bond with Ru;

(e) a —COOH group, one of whose oxygen atoms forms a bond with Ru;

(f) a —PR₂ group whose phosphorus atom forms a bond with Ru, wherein Ris independently —H, —C₁-C₁₈ alkyl, or aryl; or

(g) an —SR group whose sulfur atom forms a bond with Ru, wherein R isindependently —H, —C₁-C₁₈ alkyl, or aryl;

L² is (R⁹)₃P, (R⁹O)₃P, or L¹ and m is 2; or L² is —CN and m is 1; or L²is a labeling molecule or an active derivative thereof connected to Ruthrough the phosphorous atom of (R⁹)₂P or (R⁹O)₂P and m is 2; or L² is alabeling molecule or an active derivative thereof connected to Ruthrough the nitrogen atom of: NHR⁹, N(R⁹)₂, pyridyl, C(R⁹)═NH, C(R⁹)═NR⁹cyclic aliphatic amine group or nitrile and m is 2;

wherein each R⁹ is independently —C₁-C₁₈ alkyl, —C₃-C₈ cycloalkyl, orphenyl;

wherein when L² is P(phenyl)₃ or a labeling molecule or an activederivative thereof connected to Ru through the phosphorous atom ofP(phenyl)₂, each phenyl is independently substituted with —C₁-C₁₈ alkyl,—(C₁-C₁₈ alkyl)-OH, aryl, —(C₁-C₁₈ alkyl)-oxy, —(C₁-C₁₈ alkyl)-amino,—(C₁-C₁₈ alkyl)-thio, —CO₂Y, —C(═O)Y, —C(═O)NY₂, —NH₂, —NO₂, —OH, or—SH, and

R¹ to R⁴ are independently —H, —C₁-C₁₈ alkyl; —NH₂, —(C₁-C₁₈alkyl)-O—(C₁-C₁₈ alkyl), —OC(O)(C₁-C₁₈ alkyl), —(C₁-C₁₈ alkyl)-OH, aryl,—(C₁-C₁₈ alkyl)-oxy, —(C₁-C₁₈ alkyl)-amino, —(C₁-C₁₈ alkyl)-thio, —CO₂Y,—C(═O)Y, —C(═O)NY₂, —NO₂, or —SH, or R¹ and R² and/or R³ and R⁴ cancombine to form a carbocyclic ring substituted by one or more oxogroups;

wherein when L² is not P(phenyl)₃, R¹ to R⁴ are independently —H,—(C₁-C₁₈ alkyl)-OH, aryl, —(C₁-C₁₈ alkyl)-oxy, —(C₁-C₁₈ alkyl)-amino,—(C₁-C₁₈ alkyl)-thio, —CO₂Y, —C(═O)Y, —C(═O)NY₂, —NO₂, —OH, or —SH, orR¹ and R² and/or R³ and R⁴ can combine to form a carbocyclic ringsubstituted by one or more oxo groups, wherein at least one of R¹ to R⁴is not H;

X is Cl⁻, F⁻, Br⁻, I⁻, PF₆ ⁻, CF₃SO₃ ⁻, (C₁-C₁₈ alkyl)-CO₂ ⁻, or (C₁-C₁₈alkyl)-SO₃ ⁻; and

Y is selected from the group consisting of —H, —C₁-C₁₈ alkyl, aryl,—(C₁-C₁₈ alkyl)-aryl, —C₃-C₈ cycloalkyl, heteroaryl, and heterocyclyl.

In one embodiment of the compounds of Formula V,

each L¹ is independently an organic molecule having:

(a) a 5-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with Ru;

(b) a 6-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with Ru;

(c) an 8-10-membered bicyclic ring, one of the bicyclic rings beingaromatic and having a nitrogen atom member that forms a bond with Ru;

(d) an —NH₂ group whose nitrogen atom forms a bond with Ru;

(e) a —COOH group, one of whose oxygen atoms forms a bond with Ru;

(f) a —PR₂ group whose phosphorus atom forms a bond with Ru, wherein Ris independently —H, —C₁-C₁₈ alkyl, or aryl;

(g) an —SR group whose sulfur atom forms a bond with Ru, wherein R isindependently —H, —C₁-C₁₈ alkyl, or aryl; or

(h) a —CN group whose nitrogen atom forms a bond with Ru;

wherein when L² is P(phenyl)₃, each phenyl is independently substitutedwith —C₁-C₁₈ alkyl, —(C₁-C₁₈ alkyl)-OH, aryl, —(C₁-C₁₈ alkyl)-oxy,—(C₁-C₁₈ alkyl)-amino, —(C₁-C₁₈ alkyl)-thio, —CO₂Y, —C(═O)Y, —C(═O)NY₂,—NH₂, —NO₂, —OH, or —SH, and

R¹ to R⁴ are independently —H, —C₁-C₁₈ alkyl; —NH₂, —(C₁-C₁₈alkyl)-O—(C₁-C₁₈ alkyl), —OC(O)(C₁-C₁₈ alkyl), —(C₁-C₁₈ alkyl)-OH, aryl,—(C₁-C₁₈ alkyl)-oxy, —(C₁-C₁₈ alkyl)-amino, —(C₁-C₁₈ alkyl)-thio, —CO₂Y,—C(═O)Y, —C(═O)NY₂, —NO₂, or —SH, or R¹ and R² and/or R³ and R⁴ cancombine to form a carbocyclic ring substituted by one or more oxogroups;

wherein when L² is not P(phenyl)₃, R¹ to R⁴ are independently —H,—(C₁-C₁₈ alkyl)-OH, aryl, —(C₁-C₁₈ alkyl)-oxy, —(C₁-C₁₈ alkyl)-amino,—(C₁-C₁₈ alkyl)-thio, —CO₂Y, —C(═O)Y, —C(═O)NY₂, —NO₂, —OH, or —SH, orR¹ and R² and/or R³ and R⁴ can combine to form a carbocyclic ringsubstituted by one or more oxo groups, wherein at least one of R¹ to R⁴is not H;

X is Cl⁻, F⁻, Br⁻, I⁻, PF₆ ⁻, CF₃SO₃ ⁻, (C₁-C₁₈ alkyl)-CO₂ ⁻, or (C₁-C₁₈alkyl)-SO₃ ⁻; and

Y is selected from the group consisting of —H, —C₁-C₁₈ alkyl, aryl,—(C₁-C₁₈ alkyl)-aryl, —C₃-C₈ cycloalkyl, heteroaryl, and heterocyclyl.

In one embodiment of the compounds of Formula V,

each L¹ is independently an organic molecule having:

(a) a 5-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with Ru;

(b) a 6-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with Ru;

(c) an 8-10-membered bicyclic ring, one of the bicyclic rings beingaromatic and having a nitrogen atom member that forms a bond with Ru;

(d) an —NH₂ group whose nitrogen atom forms a bond with Ru;

(e) a —COOH group, one of whose oxygen atoms forms a bond with Ru;

(f) a —PR₂ group whose phosphorus atom forms a bond with Ru, wherein Ris independently —H, —C₁-C₁₈ alkyl, or aryl; or

(g) an —SR group whose sulfur atom forms a bond with Ru, wherein R isindependently —H, —C₁-C₁₈ alkyl, or aryl;

wherein when L² is P(phenyl)₃, each phenyl is independently substitutedwith —C₁-C₁₈ alkyl, —(C₁-C₁₈ alkyl)-OH, aryl, —(C₁-C₁₈ alkyl)-oxy,—(C₁-C₁₈ alkyl)-amino, —(C₁-C₁₈ alkyl)-thio, —CO₂Y, —C(═O)Y, —C(═O)NY₂,—NH₂, —NO₂, —OH, or —SH, and

R¹ to R⁴ are independently —H, —C₁-C₁₈ alkyl; —NH₂, —(C₁-C₁₈alkyl)-O—(C₁-C₁₈ alkyl), —OC(O)(C₁-C₁₈ alkyl), —(C₁-C₁₈ alkyl)-OH, aryl,—(C₁-C₁₈ alkyl)-oxy, —(C₁-C₁₈ alkyl)-amino, —(C₁-C₁₈ alkyl)-thio, —CO₂Y,—C(═O)Y, —C(═O)NY₂, —NO₂, or —SH, or R¹ and R² and/or R³ and R⁴ cancombine to form a carbocyclic ring substituted by one or more oxogroups;

wherein when L² is not P(phenyl)₃, R¹ to R⁴ are independently —H,—(C₁-C₁₈ alkyl)-OH, aryl, —(C₁-C₁₈ alkyl)-oxy, —(C₁-C₁₈ alkyl)-amino,—(C₁-C₁₈ alkyl)-thio, —CO₂Y, —C(═O)Y, —C(═O)NY₂, —NO₂, —OH, or —SH, orR¹ and R² and/or R³ and R⁴ can combine to form a carbocyclic ringsubstituted by one or more oxo groups, wherein at least one of R¹ to R⁴is not H;

X is Cl⁻, F⁻, Br⁻, I⁻, PF₆ ⁻, CF₃SO₃ ⁻, (C₁-C₁₈ alkyl)-CO₂ ⁻, or (C₁-C₁₈alkyl)-SO₃ ⁻; and

Y is selected from the group consisting of —H, —C₁-C₁₈ alkyl, aryl,—(C₁-C₁₈ alkyl)-aryl, —C₃-C₈ cycloalkyl, heteroaryl, and heterocyclyl.

In some embodiments of the compounds of Formula V, when L² is (R⁹)₃P,each R⁹ is different from each other.

In some embodiments of the compounds of Formula V, when L² is (R⁹)₃P,two R⁹ are the same and different from the remaining R⁹.

In some embodiments of the compounds of Formula V, when L² is (Ph)₃P,each phenyl is not substituted with methyl.

In another aspect, the present invention provides compounds of FormulaVI:

wherein:

each L¹ is independently an organic molecule having:

(a) a 5-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with Ru;

(b) a 6-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with Ru;

(c) an 8-10-membered bicyclic ring, one of the bicyclic rings beingaromatic and having a nitrogen atom member that forms a bond with Ru;

(d) an —NH₂ group whose nitrogen atom forms a bond with Ru;

(e) a —COOH group, one of whose oxygen atoms forms a bond with Ru;

(f) a —PR₂ group whose phosphorus atom forms a bond with Ru, wherein Ris independently —H, —C₁-C₁₈ alkyl, or aryl;

(g) an —SR group whose sulfur atom forms a bond with Ru, wherein R isindependently —H, —C₁-C₁₈ alkyl, or aryl; or

(h) a —CN group whose nitrogen atom forms a bond with Ru;

L² is P(phenyl)₃, wherein each phenyl is independently substituted with—C₁-C₁₈ alkyl, —(C₁-C₁₈ alkyl)-OH, aryl, —(C₁-C₁₈ alkyl)-oxy, —(C₁-C₁₈alkyl)-amino, —(C₁-C₁₈ alkyl)-thio, —CO₂Y, —C(═O)Y, —C(═O)NY₂, —NH₂,—NO₂, —OH, or —SH; or L² is a labeling molecule or an active derivativethereof connected to Ru through the phosphorous atom of (R⁹)₂P or(R⁹O)₂P and m is 2; or L² is a labeling molecule or an active derivativethereof connected to Ru through the nitrogen atom of: NHR⁹, N(R⁹)₂,pyridyl, C(R⁹)═NH, C(R⁹)═NR⁹ cyclic aliphatic amine group or nitrile andm is 2;

wherein each R⁹ is independently —C₁-C₁₈ alkyl, —C₃-C₈ cycloalkyl, orphenyl;

R¹ to R⁴ are independently —H, —C₁-C₁₈ alkyl; —NH₂, —(C₁-C₁₈alkyl)-O—(C₁-C₁₈ alkyl), —OC(O)(C₁-C₁₈ alkyl), —(C₁-C₁₈ alkyl)-OH, aryl,—(C₁-C₁₈ alkyl)-oxy, —(C₁-C₁₈ alkyl)-amino, —(C₁-C₁₈ alkyl)-thio, —CO₂Y,—C(═O)Y, —C(═O)NY₂, —NO₂, or —SH, or R¹ and R² and/or R³ and R⁴ cancombine to form a carbocyclic ring substituted by one or more oxogroups;

X is Cl⁻, F⁻, Br⁻, I⁻, PF₆ ⁻, CF₃SO₃ ⁻, (C₁-C₁₈ alkyl)-CO₂ ⁻, or (C₁-C₁₈alkyl)-SO₃ ⁻; and

Y is selected from the group consisting of —H, —C₁-C₁₈ alkyl, aryl,—(C₁-C₁₈ alkyl)-aryl, —C₃-C₈ cycloalkyl, heteroaryl, and heterocyclyl.

In one embodiment of the compounds of Formula VI:

each L¹ is independently an organic molecule having:

(a) a 5-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with Ru;

(b) a 6-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with Ru;

(c) an 8-10-membered bicyclic ring, one of the bicyclic rings beingaromatic and having a nitrogen atom member that forms a bond with Ru;

(d) an —NH₂ group whose nitrogen atom forms a bond with Ru;

(e) a —COOH group, one of whose oxygen atoms forms a bond with Ru;

(f) a —PR₂ group whose phosphorus atom forms a bond with Ru, wherein Ris independently —H, —C₁-C₁₈ alkyl, or aryl; or

(g) an —SR group whose sulfur atom forms a bond with Ru, wherein R isindependently —H, —C₁-C₁₈ alkyl, or aryl;

L² is P(phenyl)₃, wherein each phenyl is independently substituted with—C₁-C₁₈ alkyl, —(C₁-C₁₈ alkyl)-OH, aryl, —(C₁-C₁₈ alkyl)-oxy, —(C₁-C₁₈alkyl)-amino, —(C₁-C₁₈ alkyl)-thio, —CO₂Y, —C(═O)Y, —C(═O)NY₂, —NH₂,—NO₂, —OH, or —SH; or L² is a labeling molecule or an active derivativethereof connected to Ru through the phosphorous atom of (R⁹)₂P or(R⁹O)₂P and m is 2; or L² is a labeling molecule or an active derivativethereof connected to Ru through the nitrogen atom of: NHR⁹, N(R⁹)₂,pyridyl, C(R⁹)═NH, C(R⁹)═NR⁹ cyclic aliphatic amine group or nitrile andm is 2;

wherein each R⁹ is independently —C₁-C₁₈ alkyl, —C₃-C₈ cycloalkyl, orphenyl;

R¹ to R⁴ are independently —H, —C₁-C₁₈ alkyl; —NH₂, —(C₁-C₁₈alkyl)-O—(C₁-C₁₈ alkyl), —OC(O)(C₁-C₁₈ alkyl), —(C₁-C₁₈ alkyl)-OH, aryl,—(C₁-C₁₈ alkyl)-oxy, —(C₁-C₁₈ alkyl)-amino, —(C₁-C₁₈ alkyl)-thio, —CO₂Y,—C(═O)Y, —C(═O)NY₂, —NO₂, or —SH, or R¹ and R² and/or R³ and R⁴ cancombine to form a carbocyclic ring substituted by one or more oxogroups;

X is Cl⁻, F⁻, Br⁻, I⁻, PF₆ ⁻, CF₃SO₃ ⁻, (C₁-C₁₈ alkyl)-CO₂ ⁻, or (C₁-C₁₈alkyl)-SO₃ ⁻; and

Y is selected from the group consisting of —H, —C₁-C₁₈ alkyl, aryl,—(C₁-C₁₈ alkyl)-aryl, —C₃-C₈ cycloalkyl, heteroaryl, and heterocyclyl.

In one embodiment of the compounds of Formula VI,

each L¹ is independently an organic molecule having:

(a) a 5-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with Ru;

(b) a 6-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with Ru;

(c) an 8-10-membered bicyclic ring, one of the bicyclic rings beingaromatic and having a nitrogen atom member that forms a bond with Ru;

(d) an —NH₂ group whose nitrogen atom forms a bond with Ru;

(e) a —COOH group, one of whose oxygen atoms forms a bond with Ru;

(f) a —PR₂ group whose phosphorus atom forms a bond with Ru, wherein Ris independently —H, —C₁-C₁₈ alkyl, or aryl;

(g) an —SR group whose sulfur atom forms a bond with Ru, wherein R isindependently —H, —C₁-C₁₈ alkyl, or aryl; or

(h) a —CN group whose nitrogen atom forms a bond with Ru;

L² is P(phenyl)₃, wherein each phenyl is independently substituted with—C₁-C₁₈ alkyl, —(C₁-C₁₈ alkyl)-OH, aryl, —(C₁-C₁₈ alkyl)-oxy, —(C₁-C₁₈alkyl)-amino, —(C₁-C₁₈ alkyl)-thio, —CO₂Y, —C(═O)Y, —C(═O)NY₂, —NH₂,—NO₂, —OH, or —SH;

R¹ to R⁴ are independently —H, —C₁-C₁₈ alkyl; —NH₂, —(C₁-C₁₈alkyl)-O—(C₁-C₁₈ alkyl), —OC(O)(C₁-C₁₈ alkyl), —(C₁-C₁₈ alkyl)-OH, aryl,—(C₁-C₁₈ alkyl)-oxy, —(C₁-C₁₈ alkyl)-amino, —(C₁-C₁₈ alkyl)-thio, —CO₂Y,—C(═O)Y, —C(═O)NY₂, —NO₂, or —SH, or R¹ and R² and/or R³ and R⁴ cancombine to form a carbocyclic ring substituted by one or more oxogroups;

X is Cl⁻, F⁻, Br⁻, I⁻, PF₆ ⁻, CF₃SO₃ ⁻, (C₁-C₁₈ alkyl)-CO₂ ⁻, or (C₁-C₁₈alkyl)-SO₃ ⁻; and

Y is selected from the group consisting of —H, —C₁-C₁₈ alkyl, aryl,—(C₁-C₁₈ alkyl)-aryl, —C₃-C₈ cycloalkyl, heteroaryl, and heterocyclyl.

In one embodiment of the compounds of Formula VI,

each L¹ is independently an organic molecule having:

(a) a 5-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with Ru;

(b) a 6-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with Ru;

(c) an 8-10-membered bicyclic ring, one of the bicyclic rings beingaromatic and having a nitrogen atom member that forms a bond with Ru;

(d) an —NH₂ group whose nitrogen atom forms a bond with Ru;

(e) a —COOH group, one of whose oxygen atoms forms a bond with Ru;

(f) a —PR₂ group whose phosphorus atom forms a bond with Ru, wherein Ris independently —H, —C₁-C₁₈ alkyl, or aryl; or

(g) an —SR group whose sulfur atom forms a bond with Ru, wherein R isindependently —H, —C₁-C₁₈ alkyl, or aryl;

L² is P(phenyl)₃, wherein each phenyl is independently substituted with—C₁-C₁₈ alkyl, —(C₁-C₁₈ alkyl)-OH, aryl, —(C₁-C₁₈ alkyl)-oxy, —(C₁-C₁₈alkyl)-amino, —(C₁-C₁₈ alkyl)-thio, —CO₂Y, —C(═O)Y, —C(═O)NY₂, —NH₂,—NO₂, —OH, or —SH;

R¹ to R⁴ are independently —H, —C₁-C₁₈ alkyl; —NH₂, —(C₁-C₁₈alkyl)-O—(C₁-C₁₈ alkyl), —OC(O)(C₁-C₁₈ alkyl), —(C₁-C₁₈ alkyl)-OH, aryl,—(C₁-C₁₈ alkyl)-oxy, —(C₁-C₁₈ alkyl-amino, —(C₁-C₁₈ alkyl)-thio, —CO₂Y,—C(═O)Y, —C(═O)NY₂, —NO₂, or —SH, or R¹ and R² and/or R³ and R⁴ cancombine to form a carbocyclic ring substituted by one or more oxogroups;

X is Cl⁻, F⁻, Br⁻, I⁻, PF₆ ⁻, CF₃SO₃ ⁻, (C₁-C₁₈ alkyl)-CO₂ ⁻, or (C₁-C₁₈alkyl)-SO₃ ⁻; and

Y is selected from the group consisting of —H, —C₁-C₁₈ alkyl, aryl,—(C₁-C₁₈ alkyl)-aryl, —C₃-C₈ cycloalkyl, heteroaryl, and heterocyclyl.

In some embodiments of the compounds of Formula VI, when L² is (R⁹)₃P,each R⁹ is different from each other.

In some embodiments of the compounds of Formula VI, when L² is (R⁹)₃P,two R⁹ are the same and different from the remaining R⁹.

In some embodiments of the compounds of Formula VI, when L² is (Ph)₃P,each phenyl is not substituted with methyl.

In another aspect, the present invention provides compounds of FormulaVII:

wherein:

each L¹ is independently an organic molecule having:

(a) a 5-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with Ru;

(b) a 6-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with Ru;

(c) an 8-10-membered bicyclic ring, one of the bicyclic rings beingaromatic and having a nitrogen atom member that forms a bond with Ru;

(d) an —NH₂ group whose nitrogen atom forms a bond with Ru;

(e) a —COOH group, one of whose oxygen atoms forms a bond with Ru;

(f) a —PR₂ group whose phosphorus atom forms a bond with Ru, wherein Ris independently —H, —C₁-C₁₈ alkyl, or aryl;

(g) an —SR group whose sulfur atom forms a bond with Ru, wherein R isindependently —H, —C₁-C₁₈ alkyl, or aryl; or

(h) a —CN group whose nitrogen atom forms a bond with Ru;

L² is (R⁹)₃P, (R⁹O)₃P, or L¹, wherein each R⁵ is independently —C₁-C₁₈alkyl, —C₃-C₈ cycloalkyl, or phenyl, m is 2, and L² is not P(phenyl)₃;or L² is —CN and m is 1; or L² is a labeling molecule or an activederivative thereof connected to Ru through the phosphorous atom of(R⁹)₂P or (R⁹O)₂P and m is 2; or L² is a labeling molecule or an activederivative thereof connected to Ru through the nitrogen atom of: NHR⁹,N(R⁹)₂, pyridyl, C(R⁹)═NH, C(R⁹)═NR⁹ cyclic aliphatic amine group ornitrile and m is 2;

wherein each R⁹ is independently —C₁-C₁₈ alkyl, —C₃-C₈ cycloalkyl, orphenyl;

R¹ to R⁴ are independently —H, —(C₁-C₁₈ alkyl)-OH, aryl, —(C₁-C₁₈alkyl)-oxy, —(C₁-C₁₈ alkyl)-amino, —(C₁-C₁₈ alkyl)-thio, —CO₂Y, —C(═O)Y,—C(═O)NY₂, —NO₂, —OH, or —SH, or R¹ and R² and/or R³ and R⁴ can combineto form a carbocyclic ring substituted by one or more oxo groups,wherein at least one of R¹ to R⁴ is not H;

X is Cl⁻, F⁻, Br⁻, I⁻, PF₆ ⁻, CF₃SO₃ ⁻, (C₁-C₁₈ alkyl)-CO₂ ⁻, or (C₁-C₁₈alkyl)-SO₃ ⁻; and

Y is selected from the group consisting of —H, —C₁-C₁₈ alkyl, aryl,—(C₁-C₁₈ alkyl)-aryl, —C₃-C₈ cycloalkyl, heteroaryl, and heterocyclyl.

In one embodiment of the compounds of Formula VII:

each L¹ is independently an organic molecule having:

(a) a 5-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with Ru;

(b) a 6-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with Ru;

(c) an 8-10-membered bicyclic ring, one of the bicyclic rings beingaromatic and having a nitrogen atom member that forms a bond with Ru;

(d) an —NH₂ group whose nitrogen atom forms a bond with Ru;

(e) a —COOH group, one of whose oxygen atoms forms a bond with Ru;

(f), a —PR₂ group whose phosphorus atom forms a bond with Ru, wherein Ris independently —H, —C₁-C₁₈ alkyl, or aryl; or

(g) an —SR group whose sulfur atom forms a bond with Ru, wherein R isindependently —H, —C₁-C₁₈ alkyl, or aryl;

L² is (R⁹)₃P, (R⁹O)₃P, or L¹, wherein each R⁵ is independently —C₁-C₁₈alkyl, —C₃-C₈ cycloalkyl, or phenyl, m is 2, and L² is not P(phenyl)₃;or L² is —CN and m is 1; or L² is a labeling molecule or an activederivative thereof connected to Ru through the phosphorous atom of(R⁹)₂P or (R⁹O)₂P and m is 2; or L² is a labeling molecule or an activederivative thereof connected to Ru through the nitrogen atom of: NHR⁹,N(R⁹)₂, pyridyl, C(R⁹)═NH, C(R⁹)═NR⁹ cyclic aliphatic amine group ornitrile and m is 2;

wherein each R⁹ is independently —C₁-C₁₈ alkyl, —C₃-C₈ cycloalkyl, orphenyl;

R¹ to R⁴ are independently —H, —(C₁-C₁₈ alkyl)-OH, aryl, —(C₁-C₁₈alkyl)-oxy, —(C₁-C₁₈ alkyl)-amino, —(C₁-C₁₈ alkyl)-thio, —CO₂Y, —C(═O)Y,—C(═O)NY₂, —NO₂, —OH, or —SH, or R¹ and R² and/or R³ and R⁴ can combineto form a carbocyclic ring substituted by one or more oxo groups,wherein at least one of R¹ to R⁴ is not H;

X is Cl⁻, F⁻, Br⁻, I⁻, PF₆ ⁻, CF₃SO₃ ⁻, (C₁-C₁₈ alkyl)-CO₂ ⁻, or (C₁-C₁₈alkyl)-SO₃ ⁻; and

Y is selected from the group consisting of —H, —C₁-C₁₈ alkyl, aryl,—(C₁-C₁₈ alkyl)-aryl, —C₃-C₈ cycloalkyl, heteroaryl, and heterocyclyl.

In one embodiment of the compounds of Formula VII,

each L¹ is independently an organic molecule having:

(a) a 5-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with Ru;

(b) a 6-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with Ru;

(c) an 8-10-membered bicyclic ring, one of the bicyclic rings beingaromatic and having a nitrogen atom member that forms a bond with Ru;

(d) an —NH₂ group whose nitrogen atom forms a bond with Ru;

(e) a —COOH group, one of whose oxygen atoms forms a bond with Ru;

(f) a —PR₂ group whose phosphorus atom forms a bond with Ru, wherein Ris independently —H, —C₁-C₁₈ alkyl, or aryl;

(g) an —SR group whose sulfur atom forms a bond with Ru, wherein R isindependently —H, —C₁-C₁₈ alkyl, or aryl; or

(h) a —CN group whose nitrogen atom forms a bond with Ru;

L² is (R⁹)₃P, (R⁹O)₃P, or L¹, wherein each R⁵ is independently —C₁-C₁₈alkyl, —C₃-C₈ cycloalkyl, or phenyl, m is 2, and L² is not P(phenyl)₃;or L² is —CN and m is 1;

R¹ to R⁴ are independently —H, —(C₁-C₁₈ alkyl)-OH, aryl, —(C₁-C₁₈alkyl)-oxy, —(C₁-C₁₈ alkyl)-amino, —(C₁-C₁₈ alkyl)-thio, —CO₂Y, —C(═O)Y,—C(═O)NY₂, —NO₂, —OH, or —SH, or R¹ and R² and/or R³ and R⁴ can combineto form a carbocyclic ring substituted by one or more oxo groups,wherein at least one of R¹ to R⁴ is not H;

X is Cl⁻, F⁻, Br⁻, I⁻, PF₆ ⁻, CF₃SO₃ ⁻, (C₁-C₁₈ alkyl)-CO₂ ⁻, or (C₁-C₁₈alkyl)-SO₃ ⁻; and

Y is selected from the group consisting of —H, —C₁-C₁₈ alkyl, aryl,—(C₁-C₁₈ alkyl)-aryl, —C₃-C₈ cycloalkyl, heteroaryl, and heterocyclyl.

In one embodiment of the compounds of Formula VII,

each L¹ is independently an organic molecule having:

(a) a 5-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with Ru;

(b) a 6-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with Ru;

(c) an 8-10-membered bicyclic ring, one of the bicyclic rings beingaromatic and having a nitrogen atom member that forms a bond with Ru;

(d) an —NH₂ group whose nitrogen atom forms a bond with Ru;

(e) a —COOH group, one of whose oxygen atoms forms a bond with Ru;

(f) a —PR₂ group whose phosphorus atom forms a bond with Ru, wherein Ris independently —H, —C₁-C₁₈ alkyl, or aryl; or

(g) an —SR group whose sulfur atom forms a bond with Ru, wherein R isindependently —H, —C₁-C₁₈ alkyl, or aryl;

L² is (R⁹)₃P, (R⁹O)₃P, or L¹, wherein each R⁵ is independently —C₁-C₁₈alkyl, —C₃-C₈ cycloalkyl, or phenyl, m is 2, and L² is not P(phenyl)₃;or L² is —CN and m is 1;

R¹ to R⁴ are independently —H, —(C₁-C₁₈ alkyl)-OH, aryl, —(C₁-C₁₈alkyl)-oxy, —(C₁-C₁₈ alkyl)-amino, —(C₁-C₁₈ alkyl)-thio, —CO₂Y, —C(═O)Y,—C(═O)NY₂, —NO₂, —OH, or —SH, or R¹ and R² and/or R³ and R⁴ can combineto form a carbocyclic ring substituted by one or more oxo groups,wherein at least one of R¹ to R⁴ is not H;

X is Cl⁻, F⁻, Br⁻, I⁻, PF₆ ⁻, CF₃SO₃ ⁻, (C₁-C₁₈ alkyl)-CO₂ ⁻, or (C₁-C₁₈alkyl)-SO₃ ⁻; and

Y is selected from the group consisting of —H, —C₁-C₁₈ alkyl, aryl,—(C₁-C₁₈ alkyl)-aryl, —C₃-C₈ cycloalkyl, heteroaryl, and heterocyclyl.

In some embodiments of the compounds of Formula VII, when L² is (R⁹)₃P,each R⁹ is different from each other.

In some embodiments of the compounds of Formula VII, when L² is (R⁹)₃P,two R⁹ are the same and different from the remaining R⁹.

In some embodiments of the compounds of Formula VII, when L² is (Ph)₃P,each phenyl is not substituted with methyl.

In another aspect, the present invention provides compounds of FormulaVIII:

wherein:

each L¹ is independently a labeling molecule or an active derivativethereof connected to Ru through the phosphorous atom of (R⁹)₂P or(R⁹O)₂P and m is 2; or L¹ is a labeling molecule or an active derivativethereof connected to Ru through the nitrogen atom of: NHR⁹, N(R⁹)₂,pyridyl, C(R⁹)═NH, C(R⁹)═NR⁹ cyclic aliphatic amine group or nitrile andm is 2;

wherein each R⁹ is independently —C₁-C₁₈ alkyl, —C₃-C₈ cycloalkyl, orphenyl;

L² is Cl—, phosphine, OH₂, or pyridine and m is 2; or L² is —CN and m is1;

R¹ to R⁴ are independently —H, —C₁-C₁₈ alkyl; —NH₂, —(C₁-C₁₈alkyl)-O—(C₁-C₁₈ alkyl), —OC(O)(C₁-C₁₈ alkyl), —(C₁-C₁₈ alkyl)-OH, aryl,—(C₁-C₁₈ alkyl)-oxy, —(C₁-C₁₈ alkyl)-amino, —(C₁-C₁₈ alkyl)-thio, —CO₂Y,—C(═O)Y, —C(═O)NY₂, —NO₂, or —SH, or R¹ and R² and/or R³ and R⁴ cancombine to form a carbocyclic ring substituted by one or more oxogroups;

X is Cl⁻, F⁻, Br⁻, I⁻, PF₆ ⁻, CF₃SO₃ ⁻, (C₁-C₁₈ alkyl)-CO₂ ⁻, or (C₁-C₁₈alkyl)-SO₃ ⁻; and

Y is selected from the group consisting of —H, —C₁-C₁₈ alkyl, aryl,—(C₁-C₁₈ alkyl)-aryl, —C₃-C₈ cycloalkyl, heteroaryl, and heterocyclyl.

In one embodiment of the compounds of formula VIII,

each L¹ is independently a labeling molecule;

L² is Cl—, phosphine, OH₂, or pyridine and m is 2; or L² is —CN and m is1;

R¹ to R⁴ are independently —H, —C₁-C₁₈ alkyl; —NH₂, —(C₁-C₁₈alkyl)-O—(C₁-C₁₈ alkyl), —OC(O)(C₁-C₁₈ alkyl), —(C₁-C₁₈ alkyl)-OH, aryl,—(C₁-C₁₈ alkyl)-oxy, —(C₁-C₁₈ alkyl)-amino, —(C₁-C₁₈ alkyl)-thio, —CO₂Y,—C(═O)Y, —C(═O)NY₂, —NO₂, or —SH, or R¹ and R² and/or R³ and R⁴ cancombine to form a carbocyclic ring substituted by one or more oxogroups;

X is Cl⁻, F⁻, Br⁻, I⁻, PF₆ ⁻, CF₃SO₃ ⁻, (C₁-C₁₈ alkyl)-CO₂ ⁻, or (C₁-C₁₈alkyl)-SO₃ ⁻; and

Y is selected from the group consisting of —H, —C₁-C₁₈ alkyl, aryl,—(C₁-C₁₈ alkyl)-aryl, —C₃-C₈ cycloalkyl, heteroaryl, and heterocyclyl.

In some embodiments of the compounds of Formula I-III, R¹-R⁸ are eachhydrogen.

In some embodiments of the compounds of Formula V-VIII, R¹-R⁴ are eachhydrogen.

In some embodiments of the compounds of Formula VII-VIII, L¹ is methylbeta-D-1-thiogalactopyranoside. In some embodiments of the compounds ofFormula VII-VIII, L¹ is methyl beta-D-1-thiogalactopyranoside and L² is(R⁹)₃P. In some embodiments of the compounds of Formula VII-VIII, L¹ ismethyl beta-D-1-thiogalactopyranoside, L² is (Ph)₃P.

In some embodiments of the compounds of Formula VII-VIII, L¹ isisopropyl beta-D-1-thiogalactopyranoside. In some embodiments of thecompounds of Formula VII-VIII, L¹ is isopropylbeta-D-1-thiogalactopyranoside and L² is (R⁹)₃P. In some embodiments ofthe compounds of Formula VII-VIII, L¹ is isopropylbeta-D-1-thiogalactopyranoside, L² is (Ph)₃P.

In some embodiments of the compounds of Formula VIII, when L² is (R⁹)₃P,each R⁹ is different from each other.

In some embodiments of the compounds of Formula VIII, when L² is (R⁹)₃P,two R⁹ are the same and different from the remaining R⁹.

In some embodiments of the compounds of Formula VIII, when L² is (Ph)₃P,each phenyl is not substituted with methyl.

In some embodiments of the compounds of Formula I-III and V-VIII, L² is(R⁹)₃P. In some embodiments of the compounds of Formula I-III andV-VIII, L² is (R⁹)₃P, wherein each R⁹ is independently —(C₁-C₁₈)-alkyl.In some embodiments of the compounds of Formula I-III and V-VIII, L² is(R⁹)₃P, wherein each R⁹ is independently —(C₁-C₆)-alkyl. In someembodiments of the compounds of Formula I-III and V-VIII, L² is (R⁹)₃P,wherein each R⁹ is independently —(C₁-C₃)-alkyl. In some embodiments ofthe compounds of Formula I-III and V-VIII, L² is (Me)₃P.

In some embodiments of the compounds of Formula I-III and V-VIII, L² isRhodamine B-Methylaminopropionitrileamide (RhodB-MAPN). In someembodiments of the compounds of Formula I-III and V-VIII, L² isRhodamine G-Methylaminopropionitrileamide (RhodG-MAPN).

In some embodiments of the compounds of Formula I-III and V-VIII, L² is:

(RhodB-MAMePy) or a chloride salt thereof (RhodB-MAMePy-Cl).

In some embodiments of the compounds of Formula I-III and V-VIII, L² is:

(Rhod6G-MAMePy) or a chloride salt thereof (Rhod6G-MAMePy-Cl).

In some embodiments of the compounds of Formula I-III and V-VIII, thelabeling molecule is rhodamine B. In some embodiments of the compoundsof Formula I-III and V-VIII, the labeling molecule is rhodamine 6G.

In some embodiments of the compounds of Formula I-III and V-VIII, thelabeling molecule is selected from the group consisting of bodipy,dansyl, fluorescein, Texas red, cyanine dyes, pyrene, coumarins, CascadeBlue™, Pacific Blue, Marina Blue, Oregon Green,4′,6-Diamidino-2-phenylindole (DAPI), indopyra dyes, lucifer yellow,propidium iodide, porphyrins, and arginine. A compound of Formula I-VIII(“a Photolabile Compound”) releases L¹ upon exposure to light.

In another aspect, the present invention provides a compositioncomprising an effective amount of a Photolabile Compound and aphysiologically acceptable carrier, vehicle, diluent, or excipient.

In another aspect, the present invention provides a vessel containing aPhotolabile Compound.

In yet another of its aspects, the present invention provides a kitcomprising a Photolabile Compound and instructions for use.

Another aspect of the present invention provides methods for releasingan organic molecule or a labeling molecule from a Photolabile Compound,comprising exposing a Photolabile Compound to light under conditionssufficient to release the organic molecule or labeling molecule.

In yet another aspect, the present invention provides a method formaking an organic molecule bioavailable to a subject in need of theorganic molecule, comprising administering a Photolabile Compound to thesubject; and exposing the compound to light under conditions sufficientto release the organic molecule from the compound.

In another aspect, the present invention provides methods for treatingdiseases and disorders in a subject, comprising: (a) administering aPhotolabile Compound to the subject; and (b) exposing the PhotolabileCompound to light under conditions sufficient to release the organicmolecule from the Photolabile Compound. In some embodiments, thediseases and disorders treated by the methods of the invention includeneurological, neurophysiological, or neuromuscular diseases andconditions, such as epilepsy and multiple sclerosis; cancers;diaphoresis; and blood dyscrasias.

In another aspect, the present invention provides methods for assayingan organic molecule, comprising: (a) exposing a Photolabile Compound anda biological sample to light under conditions sufficient to release theorganic molecule from the Photolabile Compound, and (b) determining aneffect of the organic molecule on the biological sample.

In one aspect, the invention provides a compound selected from the groupconsisting of: [Ru(bpy)2(RhodB-MAPN)Cl]Cl, [Ru(bpy)2(Rhod6G-MAPN)Cl]Cl,[Ru(bpy)2(RhodB-MAMePy)Cl]Cl.

In another aspect, the invention provides a compound selected from thegroup consisting of: [Ru(bpy)2(RhodB-MAPN)Cl]Z,[Ru(bpy)2(Rhod6G-MAPN)Cl]Z, [Ru(bpy)2(RhodB-MAMePy)Cl]Z, wherein Z is ananion. In some embodiments, Z Cl⁻, F⁻, Br⁻ or I⁻.

Additional aspects, features and advantages afforded by the presentinvention will be apparent from the detailed description, figures, andexemplification hereinbelow.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 depicts a partial ¹H NMR spectra of [Ru(bpy)₂(4AP)₂]Cl₂, asdescribed herein (Example 1), showing the signals corresponding to the4-AP meta hydrogens. m1: in [Ru(bpy)₂(4AP)₂]²⁺; m2: in[Ru(bpy)₂(H₂O)(4AP)₂]²⁺; m3: in free ligand 4-AP.

FIG. 2 (Top) shows action potentials (spikes) recorded in a medicinalleech (Hirudo medicinalis) neuron for saline and solutions of[Ru(bpy)₃]Cl₂ and [Ru(bpy)₂(4AP)₂]Cl₂ (Bottom): Frequency of the spikes.Arrows indicate irradiation with Xe flashlamp. (Middle): Composition ofthe extracellular medium.

FIG. 3 shows cyclic voltammetry (CV) profile of [Ru(bpy)₂(4AP)₂]Cl₂ inwater. The supporting electrolyte was KNO₃ (1 M). dE/dt=100 mV/s inglassy carbon electrode.

FIGS. 4A and 4B show NMR spectra of [Ru(bpy)₂(4AP)₂]Cl₂ in D₂O before(FIG. 4A) and after (FIG. 4B) irradiation. Bruker 500 MHz.

FIG. 5 shows the UV-visible (UV-vis) spectra of Ru(bpy)₂(4AP)₂ beforeand after complete photolysis. The photoproducts after exposure to lightwere Ru(bpy)₂(4AP)(H₂O) and free 4AP. The complex did not undergo darkdecomposition for more than 20 hours. After 7 hours in the dark, theirradiated solution showed less than 4% of 4AP recombination.

FIG. 6 shows a UV-vis spectrum of the filter used for the ganglionirradiation experiments as described in Example 3.

FIGS. 7A and 7B show action potentials and frequency of the spikesobtained in studies of medicinal leech (Hirudo medicinalis) ganglia.FIG. 7A shows a recording of Retzius neuron voltage activity duringperfusion of free 4AP on the leech ganglia. [4AP]=0, 10, 20 and 50 mM.Flow rate=1 ml/min. Carrier: saline solution, as described in Example 3.FIG. 7B shows the recording of Retzius neuron activity during exposureto a 0.1 msec flash of green light through the filter of FIG. 6. Flowrate=1 ml/min. Carrier: saline solution, as described in Example 3.Pulse energy: 0.5 J.

FIGS. 8A and 8B relate to spectra changes of [Ru(bpy)₂(4AP)₂]Cl₂ duringexposure to light. FIG. 8A: spectrum changes of [Ru(bpy)₂(4AP)₂]Cl₂during irradiation with 473 nm laser light. Power: 6.39 mW continuous.Initial concentration of [Ru(bpy)₂(4AP)₂]Cl₂: 27.9 μM. A(473 nm)=0.18.FIG. 8B: Fraction of [Ru(bpy)₂(4AP)(H₂O)]²⁺ as a function of irradiationtime obtained from the spectra depicted in FIG. 8A.

FIG. 9 shows a graph of photoreleased 4AP versus pulse energy. The lightsource was pulsed Xe lamp with a bandpass filter. [Ru(bpy)₂(4AP)₂]Cl₂=44μM; Vol.=3 mL. The data were obtained from UV-vis spectra analysis.

FIG. 10 shows several two-photon fluorescence images of[Ru(bpy)₂(TzGly)(py)]Cl₂ at different excitation wavelengths.(Magnification: ˜20×).

FIG. 11 depicts a graph of total two-photon fluorescence versusexcitation wavelength of [Ru(bpy)₂(4AP)₂]Cl₂.

FIG. 12 presents a UV-vis spectrum of TzGly before and after irradiationwith 400-600 nm light.

FIGS. 13A and 13B: FIG. 13A depicts the structure of TzGly. FIG. 13Bdemonstrates the spiking of a mouse cortical neuron caused by theaddition of TzGly (1 μM) to neuron via perfusion. The measurementresults were obtained by the whole-cell patch-clamp method, as known andused in the art.

FIGS. 14A-G relate to experiments performed on neurons contacted with[Ru(bpy)₂(TzGly)(py)]Cl₂. FIGS. 14A, B and C show fluorescent-imagemicrographs of a neuron, including magnified views of dendritic spines.FIG. 14D shows the effect of laser irradiation (˜40 mW) on the spikingof a single neuron in the presence of [Ru(bpy)₂(TzGly)₂]Cl₂.Concentration of [Ru(bpy)₂(TzGly)₂]Cl₂=100 μM; Pulse length: 10 ms;Power: 40 mW; Wavelength: 720 nm. FIGS. 14E-G relate to experimentscarried out as controls to the experiments of FIGS. 14A-D. FIGS. 14E andF show magnified views of the dendritic spines of a neuron. FIG. 14Gpresents a plot showing the effect of laser irradiation on a controlneuron in the absence of [Ru(bpy)₂(TzGly)₂]Cl₂. No increased activity isobserved. Pulse length: 10 ms; Power: 40 mW; Wavelength: 720 nm.

FIG. 15 depicts a cyclic voltammetry (CV) profile of native[Ru(bpy)₂(PMe₃)Glu] at 100 mV/s on Pt wire electrode in CH₃CN containing100 mM TBAPF₆.

FIG. 16 shows (top) UV-Vis spectra of [Ru(bpy)₂(PMe₃)Glu] 0.1 mM at pH=7during 360 seconds irradiation using 450 nm light; (inset) amount ofphotoreleased glutamate derived from the experimental data (circles);fitting to the theoretical equation (line); (bottom) absorbance changesat 532 nm after flash photolysis of [Ru(bpy)₂(PMe₃)Glu] 1 mM (10ns/pulse, 256 pulses averaged).

FIG. 17 shows the aromatic section of the ¹H-NMR spectra of[Ru(bpy)₂(PMe₃)Glu] in D₂O before (upper trace) and after (lower trace)photolysis, showing the aromatics signals of [Ru(bpy)₂(PMe₃)H₂O]²⁺.

FIG. 18 shows the aliphatic section of the ¹H-NMR spectrum of[Ru(bpy)₂(PMe₃)Glu] in D₂O before (upper trace) and after (lower trace)photolysis. In the last case, the signals at 3.50, 2.30, 2.04 and 1.94ppm correspond to free glutamate.

FIG. 19 shows ¹H NMR aliphatic region spectra of the complex[Ru(bpy)₂(RhodB-MAPN)Cl]⁺ (A) before irradiation. (B) after 5 minirradiation. (C) free RhodB-MAPN spectrum. Note in B that the signals ofthe free ligand a, b, and c are apparent, indicating its photorelease.The signals d and e, corresponding to the ethyl groups in RhodB-MAPN,are far from the coordination center and therefore do not suffer bigchanges. A photolysis reaction schematic is added for clarity.

FIG. 20 shows emission spectra of a 10 μM aqueous solution of thecomplex [Ru(bpy)₂(RhodB-MAPN)Cl]⁺ during irradiation of the cuvette witha 473 nm laser diode. A spectrum was measured every 10 s. Inset showsemission maxima during irradiation.

FIG. 21 shows the UV-Vis absorption spectrum of the complex[Ru(bpy)₂(PMe₃)(MTG)]⁺² as the red line. The complex has a molarabsorptivity coefficient of ˜3100 and a maximum absorption wavelength inthe visible range at 422 nm. Photolysis quantum yield is 0.3. Successivephotolysis products of the same sample are shown in black

FIG. 22 shows a schematic drawing of [(Ru(bpy)₂P(Me)₃(MTG)]⁺². Protonsin caged MTG numbered in red correspond to signals labeled in the NMRspectrum (as indicated in FIG. 23).

FIG. 23 shows the NMR spectrum of the complex [Ru(bpy)₂(PMe₃)(MTG)]²⁺.The top trace shows the aliphatic signals of caged MTG. The middle tracecorresponds to the photolysis product. The bottom trace shows free MTG.

FIGS. 24A and B. FIG. 24A shows a handheld projector 3M (110 MPro) usedto irradiate for 20 minutes an E. coli culture growing in a piece of 7cm diameter whatman filter paper soaked in LB medium containing 1 mM[(Ru(bpy)₂P(Me)₃(MTG)]⁺² and 0.02% X-Gal. FIG. 24B shows that, afterimage projection, the culture is left at 37° C. until blue precipitateis noticed (˜40 minutes).

FIG. 25 shows the UV-Vis spectrum of the[Ru(bpy)₂(4-methylpyridine)(benzonitrile)]²⁺ complex. The spectrumcorresponding to zero photolysis is the one with the maximum absorptionpeak furthest to the left. Photolysis products have their maxima to theright of the original compound.

FIG. 26 shows the UV-Vis spectrum of the[Ru(bpy)₂(PMe₃)(3-butenenitrile)]²⁺ complex. The spectrum correspondingto zero photolysis is the one with the maximum absorption peak furthestto the left. Photolysis products have their maxima to the right of theoriginal compound.

FIG. 27 shows the UV-Vis spectrum of the[Ru(bpy)₂(PPh₃)(3-butenenitrile)]²⁺ complex. The spectrum correspondingto zero photolysis is the one with the maximum absorption peak furthestto the left. Photolysis products have their maxima to the right of theoriginal compound.

FIG. 28 shows the UV-Vis spectrum of the[Ru(bpy)₂(PMe₃)(2-cyanophenol)]²⁺ complex. The spectrum corresponding tozero photolysis is the one with the maximum absorption peak furthest tothe left. Photolysis products have their maxima to the right of theoriginal compound.

FIG. 29 shows the UV-Vis spectrum of the[Ru(bpy)₂(4-methylpyridine)(2-cyanophenol)]²⁺ complex. The spectrumcorresponding to zero photolysis is the one with the maximum absorptionpeak furthest to the left. Photolysis products have their maxima to theright of the original compound.

FIG. 30 shows the UV-Vis spectrum of the[Ru(bpy)₂(4-methylpyridine)(3-butenenitrile)]²⁺ complex. The spectrumcorresponding to zero photolysis is the one with the maximum absorptionpeak furthest to the left. Photolysis products have their maxima to theright of the original compound.

FIG. 31 shows the UV-Vis spectrum of the[Ru(bpy)₂(PPh₃)(2-cyanophenol)]²⁺ complex. The spectrum corresponding tozero photolysis is the one with the maximum absorption peak furthest tothe left. Photolysis products have their maxima to the right of theoriginal compound.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates generally to Photolabile Compoundscomprising organic molecules or labeling molecules and methods for usingthe Photolabile Compounds. The organic molecules can be biologicallyactive. In one embodiment of this invention, an organic molecule, e.g.,a biologically active molecule, is protected and subsequently releasedupon exposure to light, advantageously, visible light.

In contrast to known methods, visible light, e.g., a visible lightpulse, can be used to release an organic molecule or a labeling moleculefrom a Photolabile Compound. Thus, in the present methods, samples,e.g., organs, tissues or cells, or subjects to which a PhotolabileCompound is administered, undergo only minimal, if any, exposure to UVradiation, which has detrimental effects on cellular components and,ultimately, on cell growth and viability.

In accordance with the present invention, and without wishing to bebound by theory, the Ru-organic molecule bond or Ru-labeling moleculebond is normally weaker than a covalent σ bond, and therefore can bebroken using a lower energy irradiation. Further in accordance with thisinvention, and without wishing to be bound by theory, the energyrequired for the release of an organic molecule or a labeling moleculeby exposure to light is relatively low. In the Photolabile Compounds ofthis invention, the organic molecule or labeling molecule isphotoreleased by irradiation of the Photolabile Compound using light asdescribed herein.

Also, according to this invention, photorelease can occur in vivo or ina biological sample, e.g., a body fluid, a body sample, such as an organor tissue sample, in living cells and in the body. Thus, the PhotolabileCompounds are especially valuable for in vivo biological applications,such as treatments for various diseases, conditions and disorders of thebody. The use of Photolabile Compounds as described herein allowsprecise control of the onset of a bioactive function or a bioactivity inthe body, for example, in living organs, tissues, and cells, i.e.,within nanoseconds to milliseconds, with minimal harm to a biologicalsample, or to the body or its organ, tissue and cellular components. Inaddition, exposure of a biological sample to light can be localized tothe site where an organic molecule is needed or desired. This isparticularly beneficial for administration to a subject, particularly ahuman patient.

The Photolabile Compounds are also suitable for use in non-biologicalsystems, such as in solar cells, photocells, or an optical memory, e.g.,a three dimensional optical memory.

In one embodiment, the invention encompasses a compound of Formula I:

wherein:

R¹-R⁸, L¹, L², X, M and m are as defined above for the compounds ofFormula I.

In another embodiment, the invention encompasses a compound of FormulaII:

wherein:

R¹-R⁸, L¹, L², X and m are as defined above for the compounds of FormulaII.

In another embodiment, the invention encompasses a compound of FormulaIII:

wherein:

R¹-R⁸, L¹, L², X and m are as defined above for the compounds of FormulaIII.

In another embodiment, the invention encompasses a compound of FormulaIV:

wherein M¹, M², and L¹ are as defined above for the compounds of FormulaIV.

In one embodiment, the invention encompasses compounds of Formula V:

wherein:

R¹-R⁴, L¹, L², X and m are as defined above for the compounds of FormulaV.

In another embodiment, the invention encompasses a compound of FormulaVI:

wherein:

R¹-R⁴, L¹, L², X and m are as defined above for the compounds of FormulaVI.

In an embodiment, the invention encompasses a compound of Formula VI,wherein L² is P(phenyl)₃ and each phenyl is independently substituted atthe 3 or 4 position.

In another embodiment, the invention encompasses a compound of FormulaVI, wherein L² is P(phenyl)₃ and at least one phenyl is substituted with—(C₁-C₁₈ alkyl)-OH.

In another embodiment, the invention encompasses a compound of FormulaVI, wherein L² is P(phenyl)₃ and each phenyl is substituted with—(C₁-C₁₈ alkyl)-OH.

In another embodiment, the invention encompasses a compound of FormulaVI, wherein L² is P(phenyl)₃ and at least one phenyl is substituted with—COOH.

In another embodiment, the invention encompasses a compound of FormulaII, wherein L² is P(phenyl)₃ and each phenyl is substituted with —COOH.

In another embodiment, the invention encompasses a compound of FormulaVI, wherein L² is P(phenyl)₃ and at least one phenyl is substituted with—OH.

In another embodiment, the invention encompasses a compound of FormulaVI, wherein L² is P(phenyl)₃ and each phenyl is substituted with —OH.

In another embodiment, the invention encompasses a compound of FormulaVI, wherein L² is P(phenyl)₃ and at least one phenyl is substituted with—NH₂.

In another embodiment, the invention encompasses a compound of FormulaVI, wherein L² is P(phenyl)₃ and each phenyl is substituted with —NH₂.

In another embodiment, the invention encompasses a compound of FormulaVI, wherein L² is P(phenyl)₃ and at least one phenyl is substituted with—NO₂.

In another embodiment, the invention encompasses a compound of FormulaVI, wherein L² is P(phenyl)₃ and each phenyl is substituted with —NO₂.

In another embodiment, the invention encompasses a compound of FormulaVI, wherein L¹ is an organic molecule comprising PMe₂ whose phosphorusatom forms a bond with Ru.

In another embodiment, the invention encompasses a compound of FormulaVI, wherein L¹ is an organic molecule comprising P(phenyl)₂ whosephosphorus atom forms a bond with Ru.

In another embodiment, the invention encompasses a compound of FormulaVII:

wherein:

R¹-R⁴, L¹, L², X and m are as defined above for the compounds of FormulaVII.

In another embodiment, the invention encompasses a compound of FormulaVII, wherein L² is P(methyl)(phenyl)₂.

In another embodiment, the invention encompasses a compound of FormulaVII, wherein L² is P(methyl)₂(phenyl).

In another embodiment, the invention encompasses a compound of FormulaVII, wherein L¹ is an organic molecule comprising PMe₂ whose phosphorusatom forms a bond with Ru.

In another embodiment, the invention encompasses a compound of FormulaVII, wherein L¹ is an organic molecule comprising P(phenyl)₂ whosephosphorus atom forms a bond with Ru.

In another embodiment, the invention encompasses a compound of FormulaVIII:

wherein:

R¹-R⁴, L¹, L², X and m are as defined above for the compounds of FormulaVIII.

The Photolabile Compounds of Formulas I-VIII can exist in a cis or transconfiguration. Accordingly, Formulas I-VIII encompass both cis and transforms of the Photolabile Compounds.

In the compounds of the present invention, the term “—(C₁-C₁₈) alkyl”refers to a saturated straight or branched non-cyclic hydrocarbon having1 to 18 carbon atoms. Representative saturated straight chain —(C₁-C₁₈)alkyls include -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl,-n-hexyl, -n-heptyl, -n-octyl, -n-nonyl, -n-decyl, -n-undecyl,-n-dodecyl, -n- tridecyl, -n-tetradecyl, -n-pentadecyl, -n-hexadecyl,-n-heptadecyl and -n-octadecyl. Representative saturated branched—(C₁-C₁₈) alkyls include -isopropyl, -sec-butyl, -isobutyl, -tert-butyl,-isopentyl, -2-methylbutyl, -3-methylbutyl, -2,2-dimethylbutyl,-2,3-dimethylbutyl, -2-methylpentyl, -3-methylpentyl, -4-methylpentyl,-2-methylhexyl, -3-methylhexyl, -4-methylhexyl, -5-methylhexyl,-2,3-dimethylbutyl, -2,3-dimethylpentyl, -2,4-dimethylpentyl,-2,2-dimethylhexyl, -2,3-dimethylhexyl, -2,4-dimethylhexyl,-2,5-dimethylhexyl, -2,2-dimethylpentyl, -3,3-dimethylpentyl,-3,3-dimethylhexyl, -4,4-dimethylhexyl, -2-ethylpentyl, -3-ethylpentyl,-2-ethylhexyl, -3-ethylhexyl, -4-ethylhexyl, -2-methyl-2-ethylpentyl,-2-methyl-3-ethylpentyl, -2-methyl-4-ethylpentyl,-2-methyl-2-ethylhexyl, -2-methyl-3-ethylhexyl, -2-methyl-4-ethylhexyl,-2,2-dimethylpentyl, -3,3-diethylhexyl, -2,2-diethylhexyl,-3,3-diethylhexyl and the like.

In the compounds of the present invention, the term “—(C₃-C₈)cycloalkyl” refers to a saturated cyclic hydrocarbon having from 3 to 8carbon atoms. Representative —(C₃-C₈) cycloalkyls include -cyclopropyl,-cyclobutyl, -cyclopentyl, -cyclohexyl, -cycloheptyl and -cyclooctylhydrocarbons.

In the compounds of the present invention, the term “aryl” refers to anaromatic group containing 1 to 3 aromatic rings, either fused or linked.

In the compounds of the present invention, the term “heterocyclic group”or “heterocyclic” or “heterocyclyl” or “heterocyclo” as used hereinrefers to fully saturated, or partially or fully unsaturated, includingaromatic (i.e., “heteroaryl”) cyclic groups (for example, 4 to 7membered monocyclic, 7 to 11 membered bicyclic, or 10 to 16 memberedtricyclic ring systems) which have at least one heteroatom in at leastone carbon atom-containing ring. Each ring of the heterocyclic groupcontaining a heteroatom may have 1, 2, 3, or 4 heteroatoms selected fromnitrogen atoms, oxygen atoms and/or sulfur atoms, where the nitrogen andsulfur heteroatoms may optionally be oxidized and the nitrogenheteroatoms may optionally be quaternized. The heterocyclic group may beattached to the remainder of the molecule at any heteroatom or carbonatom of the ring or ring system. Exemplary heterocyclic groups include,but are not limited to, azepanyl, azetidinyl, aziridinyl, dioxolanyl,furanyl, furazanyl, homo piperazinyl, imidazolidinyl, imidazolinyl,isothiazolyl, isoxazolyl, morpholinyl, oxadiazolyl, oxazolidinyl,oxazolyl, oxazolidinyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl,piperazinyl, piperidinyl, pyranyl, pyrazinyl, pyrazolidinyl,pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazolyl, pyridoimidazolyl,pyridothiazolyl, pyridinyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl,quinuclidinyl, tetrahydrofuranyl, thiadiazinyl, thiadiazolyl, thienyl,thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiomorpholinyl,thiophenyl, triazinyl, and triazolyl. Exemplary bicyclic heterocyclicgroups include indolyl, isoindolyl, benzothiazolyl, benzoxazolyl,benzoxadiazolyl, benzothienyl, quinuclidinyl, quinolinyl,tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl,indolizinyl, benzofuryl, benzofurazanyl, chromonyl, coumarinyl,benzopyranyl, cinnolinyl, quinoxalinyl, indazolyl, pyrrolopyridyl,furopyridinyl (such as furo[2,3-c]pyridinyl, furo[3,2-b]pyridinyl] orfuro[2,3-b]pyridinyl), dihydroisoindolyl, dihydroquinazolinyl (such as3,4-dihydro-4-oxo-quinazolinyl), triazinylazepinyl, tetrahydroquinolinyland the like. Exemplary tricyclic heterocyclic groups includecarbazolyl, benzidolyl, phenanthrolinyl, acridinyl, phenanthridinyl,xanthenyl and the like.

The term “cyclic aliphatic amine” or “cyclic aliphatic amine group” asused herein refers to a non-aromatic secondary amine or tertiary cyclicamine group. Examples of cyclic aliphatic amines include, but are notlimited to, aziridine and piperidine.

The term “active derivative” or “active derivative of a labelingmolecule” as used herein refers to a chemical derivative of a labelingmolecule, which retains the labeling function of the labeling molecule(e.g., its fluorescence properties). In some embodiments, such activederivative offers superior properties for attachment to Ru atom througha phosphine group, an aliphatic amine group, an imine group, a pyridylgroup, or a nitrile group. A specific example of an active derivative ofrhodamine (a labeling molecule) is B-Methylaminopropionitrileamide(RhodB-MAPN). A person of skill in the art can modify the labelingmolecule to create a derivative thereof and then test the resultantderivative for activity in the same way that the activity of a labelingmolecule is tested to determine whether the derivative is active.

An amino acid group, such as an α-amino acid, is an organic moleculehaving an amino group (—NH₂) and a carboxylic acid group. An amino acidcan be one of the 20 common α-amino acids (Gly, Ala, Val, Leu, Ile, Ser,Thr, Asp, Asn, Lys, Glu, Gln, Arg, His, Phe, Cys, Trp, Tyr, Met andPro), or another naturally occurring amino acid, such as norleucine,ethylglycine, ornithine, gamma-amino butyric acid, and phenylglycine.Other representative amino acids includeα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA).

Examples of a 6-membered monocyclic aromatic ring, wherein one of thering's members is a nitrogen atom, include a pyridyl, pyrimidinyl,pyridazinyl and pyrazinyl ring.

Examples of a 5-membered monocyclic aromatic ring, wherein one of thering's members is a nitrogen atom, include a pyrrolyl, imidazolyl,pyrazolyl, triazolyl, tetrazolyl, oxazolyl, oxadiazolyl, thiazolyl andthiadiazolyl ring.

Examples of an 8-10-membered bicyclic aromatic ring, wherein one of therings is aromatic and has a nitrogen atom member, include anindolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl,quinolyl, isoquinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl,1,3,7-trimethyl-2,6-dioxopurinyl, quinazolinyl, cinnolinyl, pteridinyl,6-amino-1H-purinyl, 2-aminohypoxanthinyl, mercaptopurine, thioguanine,and temozolomide bicyclic aromatic ring.

In general terms, illustrative examples of organic molecules useful inthe present Photolabile Compounds embrace a variety of agents, such aspharmaceutical agents, small molecules, drugs, neurochemicals, peptides,proteins, and chemotherapeutic agents, as nonlimiting examples.

Illustrative organic molecules can further include amino acids,luciferin, enzyme inhibitors, fatty acids (e.g., arachidonic acid),protein kinase C activators (e.g., dioctanoylglycerol), tubulin assemblypromoters (e.g., paclitaxel), antibiotics (e.g., penicillins or A23187),neurotransmitters (e.g., L-glutamic acid, aspartic acid,carbamylcholine, dopamine, epinephrine, GABA, glutamic acid, glycine,haloperidol, isoproterenol, kainic acid, NMDA, NMDA receptor antagonistMK-801, norepinephrine, phenylephrine, propranolol), 4-aminopyridine(4AP), serotonin (5 hydroxytriptamine, 5HT), (RS)-(tetrazol-5-yl)glycine (TzGly), α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid(AMPA), tetrazolyl-α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid((tetrazol-5-yl) AMPA), nicotine, nicotinic acid, isoxazole, andfluorescent dyes (e.g., fluorescein, HPTS, rhodamines, succinimidylesters and sulfosuccinimidyl esters of carboxy-Q-rhodamine, or RhodamineGreen), nucleotides (e.g., ATP, ADP, cAMP, GDP, GTP, cGMP, GTP-γ-S,GDP-β, 8-substituted derivatives of cAMP or cGMP, e.g., 8-bromo-cAMP,8-bromo-cGMP, 8-chloro-cAMP, 8-chloro-cGMP, 8-parachlorophenylthio(cCPT) cAMP or cGMP, phosphates (e.g., phosphates, phosphate esters),phenylphosphate (PPh₃), Py, nucleosides, nucleoside derivatives,nucleotide derivatives (e.g., cADP-ribose, 8-amino-cADP ribose, or8-bromo-cADP-ribose), cyclitols (e.g., inositol), cyclitol phosphates(e.g., myo-inositol phosphate, myo-inositol-1,4,5-triphosphate,myo-inositol-1,3,4,5-tetrakisphosphate, ormyo-inositol-3,4,5,6-tetrakisphosphate), NO (e.g., from the decomposablecompound HON═N(O) (Net₂)), chelants (e.g., EDTA, EGTA), ionophores(e.g., nigericin), alkylthiogalactose derivatives (e.g.,methylthiogalactose, isopropylthiogalactose), mercaptopurine,thioguanine, doxorubicin, cytarabin, temozolomide, gentamicin, andnitrile containing compounds. The organic molecule can be cell permeant,as described, for example, in Furuta et al., Biochem. Biophys. Res.Commun., 228:193-198 (1996).

Other useful examples of organic molecules include adenosine5′-diphosphate ADP; adenosine 5′-triphosphate ATP; adenosine5′-monophosphate AMP; aminobutyric acid; L-glutamic acid; cyclicadenosine 5′-diphosphate ribose; adenosine 3′,5′-cyclicmonophosphate;fluorescein; methyl-D-aspartic acid; tyramine; tryptophan;4-aminopyridine (4AP); epinephrine; norepinephrine; dopamine; serotonin(5 hydroxytriptamine, 5HT); (RS)-(tetrazol-5-yl) glycine (TzGly), whichis a potent N-methyl-D-aspartate receptor (NMDA) agonist;tetrazolyl-α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid((tetrazol-5-yl) AMPA); caffeine and nicotine.

In accordance with this invention, the organic molecule ligandsglutamate, gamma aminobutyric acid (GABA), alaninate, glycinate and thelike have been demonstrated to photorelease from a Photolabile Compoundafter exposure to visible light; such Photolabile Compounds are stablein solutions in addition to water. Organic molecules having an —NH₂group or an —COOH group may be released in solvents other than water,for example, alcohol (e.g., methanol, ethanol), acetone, etc.

The organic molecule of the invention include phosphorus derivativeswith a —PR₂ group whose phosphorus atom forms a bond with Ru, wherein Ris independently —H, —C₁-C₁₈ alkyl, or aryl. Exemplary phosphorusderivatives include dimethylphosphinyl, diethylphosphinyl, anddiphenylphosphinyl.

The organic molecule of the invention include sulfur derivatives with an—SR group whose sulfur atom forms a bond with Ru, wherein R isindependently —H, —C₁-C₁₈ alkyl, or aryl. Exemplary sulfur derivativesinclude methyl beta-D-1-thiogalactopyranoside (MTG), isopropylbeta-D-1-thiogalactopyranoside (IPTG), thioethers, thiolates,methylthio, ethylthio, and phenylthio.

The organic molecule of the invention include nitrile derivatives witha-CN group whose nitrogen atom forms a bond with Ru. Exemplary nitrilederivatives include alkyl nitriles, alkenyl nitriles (such as3-butenenitrile), and aryl nitriles (such as benzonitrile or2-cyanophenol). Other therapeutic agents containing nitrile groups canalso be used as organic molecules.

In general terms, illustrative examples of labeling molecules useful inthe present Photolabile Compounds embrace a variety of agents, such asmolecules with a fluorescent, a bioluminescent, a chemiluminescent, acolorimetric or a radioactive group, as nonlimiting examples.

A fluorescent molecule can be selected from bodipy, dansyl, fluorescein,rhodamine, Texas red, cyanine dyes, pyrene, coumarins, Cascade Blue™,Pacific Blue, Marina Blue, Oregon Green, 4′,6-Diamidino-2-phenyl indole(DAPI), indopyra dyes, lucifer yellow, propidium iodide, porphyrins,arginine, and variants and derivatives thereof. For further informationon fluorescent label moieties and fluorescence techniques, see, e.g.,Handbook of Fluorescent Probes and Research Chemicals, by Richard P.Haughland, Sixth Edition, Molecular Probes, (1996), which is herebyincorporated by reference in its entirety.

The Photolabile Compounds of Formulas I-III and V-VIII where L₂ is otherthan L₁ can be made by allowing about a molar equivalent of Ru(bdt)₂Cl₂,where bdt is bipyridine or phenanthroline substituted with an R₁-R₈group as defined in Formulas I-III or R₁-R₄ group as defined in FormulasV-VIII, to react with about a molar equivalent of an organic or labelingmolecule in water, ethanol, methanol, isopropyl alcohol, ethyleneglycol, acetone, methylene chloride or a mixture thereof at reflux undernitrogen. After about 4 to about 8 hours, the resultant solution iscooled and to it is added at least about an equivalent of L₂. Theresultant mixture is heated at reflux for about 4 to about 20 h. Aftercooling to room temperature, the resultant solution is diluted withwater, and to it is added excess NH₄PF₆. The resultant precipitate isfiltered, purified via silica-gel chromatography, dried and dissolved inacetone. (n-Bu)₄NH₄ ⁺ X, wherein X is defined in Formula I-III andV-VIII, is added to the acetone solution, and the resultant PhotolabileCompound of Formula I-III and V-VIII where L₂ is other than L₁ isfiltered.

The Photolabile Compounds of Formulas I-III and V-VIII where L₂ is L₁can be made by allowing about a molar equivalent of Ru(bdt)₂Cl₂, wherebdt is bipyridine or phenanthroline substituted with an R₁-R₈ group asdefined in Formulas I-IIII or R₁-R₄ group as defined in Formulas V-VIII,to react with an excess amount of an organic or labeling molecule inwater, ethanol, methanol, isopropyl alcohol, ethylene glycol, acetone,methylene chloride or a mixture thereof at reflux under nitrogen. Afterabout 4 to about 8 hours, the resultant solution is cooled to roomtemperature. The resultant mixture is diluted with water, and to it isadded excess NH₄PF₆. The resultant precipitate is filtered, purified viasilica-gel chromatography, dried and dissolved in acetone. (n-Bu)₄NH₄ ⁺X, wherein X is defined in I-III and V-VIII, is added to the acetonesolution, and the resultant Photolabile Compound of Formula I-III andV-VIII where L₂ is L₁ is filtered.

The Photolabile Compounds of Formula IV where M² is Ru can be obtainedby dissolving about 1 molar equivalent of (M¹)₃[M²(CN)₅NH₃] 2H₂O, whereM¹ is defined in Formula IV, in about 15 mL of argon-deoxygenated 1:1ethanol:water containing about 10 molar equivalents of the organicmolecule. The resultant mixture is maintained at about room temperatureunder argon for about 1 hour and concentrated in vacuo at about roomtemperature to a volume of about 1 mL. To the resultant concentrate isadded a cold, saturated ethanol solution of M¹I, resulting in aprecipitation of the Photolabile Compounds of Formula IV where M² is Ru,which are washed with ethanol and diethyl ether.

For the present invention, photorelease can generally occur rapidly,e.g., after about a few nanoseconds to about 500 milliseconds (SeeSalierno et al., J Inorg Biochem. 2010 104(4):418-22) following exposureto visible light of the appropriate wavelength. Suitable wavelengths oflight for effective photorelease of an organic molecule or a labelingmolecule from a Photolabile Compound range from about 300 to about 500nm, or from about 300 to about 360 nm, or from about 450 to about 500nm, e.g., 473 nm and may be extended up to 700 nm. Suitable lightsources include those which are capable of irradiating light of theappropriate wavelengths, for example and without limitation,commercially available tungsten lamps (Cole-Parmer), arc lamps, xenoncontinuous lamps, lasers, e.g., blue or green lasers or photooptic lightsources. Such light sources are commercially available (CrystaLaser,Reno, Nev.; Lasever, Jiangdong, Ningbo, China). Other forms of light,such as sunlight, infrared light, pulsed infrared light, or UV radiationcan also be used for the invention, as necessary or desired.

Devices and systems suitable for exposing the Photolabile Compounds tolight, particularly visible or infrared light, further include imagingprobes, imaging catheters and fiber optic probes, particularly thosecontaining gradient index, or graded-index, (GRIN) lenses, which aredescribed in U. Utzinger et al., 2003, J. Biomed. Optics, 8(1):121-147;and Fujimoto et al., Photonic Materials, Devices and Systems—LaserMedicine and Medical Imaging Group, RLE Progress Report 144, pp 27-1 to27-35, and which are commercially available. (Sp3 plus, UK). The lightsuitable for exposing the Photolabile Compounds to photorelease anorganic molecule or a labeling molecule comprises a wavelength of about300 to about 500 nm, or about 300 to about 360 nm, or about 450 to about500 nm. Suitable light includes visible or infrared light.

Further in accordance with this invention, the organic molecules orlabeling molecules can also be released from the Photolabile Compoundsvia one-photon or two-photon photolysis. Optical memories that utilize atwo-photon excitation are described, for example, by Strickler and Webb,1991, Optics Letters, 16:1780-1782. A feature of two-photon excitationis the elimination of out-of-focus background. (See, e.g., W. Denk etal., 1990, Science, 248:73-76). Thus, two-photon uncaging can release anorganic molecule or a labeling molecule only in the plane of focus.(See, e.g., W. Denk et al., 1994, Proc. Natl. Acad. Sci. USA,91:6629-6633).

In an embodiment, the present invention encompasses a compound ofFormula I, wherein the organic molecule is 4-AP. In another embodiment,the invention encompasses a compound of Formula I, wherein the organicmolecule is TzGly. In another embodiment, the invention encompasses acompound of Formula I, wherein the organic molecule is (tetrazol-5-yl)AMPA. In another embodiment, the invention encompasses a compound ofFormula I, wherein the organic molecule is nicotine or caffeine. Inanother embodiment, the invention encompasses a compound of Formula I,wherein the organic molecule is serotonin, epinephrine, norepinephrine,or dopamine. In another embodiment, the present invention encompasses acompound of Formula I, wherein the organic molecule is adenosine5′-diphosphate ADP, adenosine 5′-triphosphate ATP, adenosine5′-monophosphate AMP, cyclic adenosine 5′-diphosphate ribose, oradenosine 3′,5′-cyclicmonophosphate. In another embodiment, theinvention encompasses a compound of Formula I wherein the organicmolecule is aminobutyric acid or L-glutamic acid, or methyl-D-asparticacid. In another embodiment, the invention encompasses a compound ofFormula I wherein the organic molecule is methionine,methylthiogalactose or isopropylthiogalactose. In another embodiment,the invention encompasses a compound of Formula I wherein the organicmolecule is methylthiogalactose or isopropylthiogalactose. In anotherembodiment, the invention encompasses a compound of Formula I whereinthe organic molecule is mercaptopurine, thioguanine, doxorubicin,cytarabin, temozolomide, or gentamicin. In another embodiment, theinvention encompasses a compound of Formula I wherein the organicmolecule is mercaptopurine. In another embodiment, the inventionencompasses a compound of Formula I wherein the organic molecule isthioguanine. In another embodiment, the invention encompasses a compoundof Formula I wherein the organic molecule is doxorubicin. In anotherembodiment, the invention encompasses a compound of Formula I whereinthe organic molecule is cytarabin. In another embodiment, the inventionencompasses a compound of Formula I wherein the organic molecule istemozolomide. In another embodiment, the invention encompasses acompound of Formula I wherein the organic molecule is gentamicin. Inanother embodiment, the invention encompasses a compound of Formula Iwherein the organic molecule is benzonitrile, 2-cyanophenol or3-butenenitrile.

In an embodiment, the present invention encompasses a compound ofFormula II or III, wherein the organic molecule is 4-AP. In anotherembodiment, the invention encompasses a compound of Formula II, whereinthe organic molecule is TzGly. In another embodiment, the inventionencompasses a compound of Formula II, wherein the organic molecule is(tetrazol-5-yl) AMPA. In another embodiment, the invention encompasses acompound of Formula II, wherein the organic molecule is nicotine orcaffeine. In another embodiment, the invention encompasses a compound ofFormula II, wherein the organic molecule is serotonin, epinephrine,norepinephrine, or dopamine. In another embodiment, the presentinvention encompasses a compound of Formula II, wherein the organicmolecule is adenosine 5′-diphosphate ADP, adenosine 5′-triphosphate ATP,adenosine 5′-monophosphate AMP, cyclic adenosine 5′-diphosphate ribose,or adenosine 3′,5′-cyclicmonophosphate. In another embodiment, theinvention encompasses a compound of Formula II, wherein the organicmolecule is aminobutyric acid or L-glutamic acid, or methyl-D-asparticacid. In another embodiment, the invention encompasses a compound ofFormula II wherein the organic molecule is methionine,methylthiogalactose or isopropylthiogalactose. In another embodiment,the invention encompasses a compound of Formula II wherein the organicmolecule is methylthiogalactose or isopropylthiogalactose. In anotherembodiment, the invention encompasses a compound of Formula II whereinthe organic molecule is mercaptopurine, thioguanine, doxorubicin,cytarabin, temozolomide, or gentamicin. In another embodiment, theinvention encompasses a compound of Formula II wherein the organicmolecule is mercaptopurine. In another embodiment, the inventionencompasses a compound of Formula II wherein the organic molecule isthioguanine. In another embodiment, the invention encompasses a compoundof Formula II wherein the organic molecule is doxorubicin. In anotherembodiment, the invention encompasses a compound of Formula II whereinthe organic molecule is cytarabin. In another embodiment, the inventionencompasses a compound of Formula II wherein the organic molecule istemozolomide. In another embodiment, the invention encompasses acompound of Formula II wherein the organic molecule is gentamicin. Inanother embodiment, the invention encompasses a compound of Formula IIwherein the organic molecule is benzonitrile, 2-cyanophenol or3-butenenitrile.

In an embodiment, the present invention encompasses a compound ofFormula IV, wherein the organic molecule is 4-AP. In another embodiment,the invention encompasses a compound of Formula IV, wherein the organicmolecule TzGly. In another embodiment, the invention encompasses acompound of Formula IV, wherein the organic molecule is (tetrazol-5-yl)AMPA. In another embodiment, the invention encompasses a compound ofFormula IV, wherein the organic molecule is nicotine or caffeine. Inanother embodiment, the invention encompasses a compound of Formula IV,wherein the organic molecule is serotonin, epinephrine, norepinephrine,or dopamine. In another embodiment, the present invention encompasses acompound of Formula IV, wherein the organic molecule is adenosine5′-diphosphate ADP, adenosine 5′-triphosphate ATP, adenosine5′-monophosphate AMP, cyclic adenosine 5′-diphosphate ribose, oradenosine 3′,5′-cyclicmonophosphate. In another embodiment, theinvention encompasses a compound of Formula IV, wherein the organicmolecule is aminobutyric acid or L-glutamic acid, or methyl-D-asparticacid. In another embodiment, the invention encompasses a compound ofFormula IV wherein the organic molecule is methionine,methylthiogalactose or isopropylthiogalactose. In another embodiment,the invention encompasses a compound of Formula IV wherein the organicmolecule is methylthiogalactose or isopropylthiogalactose. In anotherembodiment, the invention encompasses a compound of Formula IV whereinthe organic molecule is mercaptopurine, thioguanine, doxorubicin,cytarabin, temozolomide, or gentamicin. In another embodiment, theinvention encompasses a compound of Formula IV wherein the organicmolecule is mercaptopurine. In another embodiment, the inventionencompasses a compound of Formula IV wherein the organic molecule isthioguanine. In another embodiment, the invention encompasses a compoundof Formula IV wherein the organic molecule is doxorubicin. In anotherembodiment, the invention encompasses a compound of Formula IV whereinthe organic molecule is cytarabin. In another embodiment, the inventionencompasses a compound of Formula IV wherein the organic molecule istemozolomide. In another embodiment, the invention encompasses acompound of Formula IV wherein the organic molecule is gentamicin. Inanother embodiment, the invention encompasses a compound of Formula IVwherein the organic molecule is benzonitrile, 2-cyanophenol or3-butenenitrile.

In an embodiment, the present invention encompasses a compound ofFormula V, wherein the organic molecule 4-AP. In another embodiment, theinvention encompasses a compound of Formula V, wherein the organicmolecule is TzGly. In another embodiment, the invention encompasses acompound of Formula V, wherein the organic molecule is (tetrazol-5-yl)AMPA. In another embodiment, the invention encompasses a compound ofFormula V, wherein the organic molecule is nicotine or caffeine. Inanother embodiment, the invention encompasses a compound of Formula V,wherein the organic molecule is serotonin, epinephrine, norepinephrine,or dopamine. In another embodiment, the present invention encompasses acompound of Formula V, wherein the organic molecule is adenosine5′-diphosphate ADP, adenosine 5′-triphosphate ATP, adenosine5′-monophosphate AMP, cyclic adenosine 5′-diphosphate ribose, oradenosine 3′,5′-cyclicmonophosphate. In another embodiment, theinvention encompasses a compound of Formula V wherein the organicmolecule is aminobutyric acid or L-glutamic acid, or methyl-D-asparticacid. In another embodiment, the invention encompasses a compound ofFormula V wherein the organic molecule is methionine,methylthiogalactose or isopropylthiogalactose. In another embodiment,the invention encompasses a compound of Formula V wherein the organicmolecule is methylthiogalactose or isopropylthiogalactose. In anotherembodiment, the invention encompasses a compound of Formula V whereinthe organic molecule is mercaptopurine, thioguanine, doxorubicin,cytarabin, temozolomide, or gentamicin. In another embodiment, theinvention encompasses a compound of Formula V wherein the organicmolecule is mercaptopurine. In another embodiment, the inventionencompasses a compound of Formula V wherein the organic molecule isthioguanine. In another embodiment, the invention encompasses a compoundof Formula V wherein the organic molecule is doxorubicin. In anotherembodiment, the invention encompasses a compound of Formula V whereinthe organic molecule is cytarabin. In another embodiment, the inventionencompasses a compound of Formula V wherein the organic molecule istemozolomide. In another embodiment, the invention encompasses acompound of Formula V wherein the organic molecule is gentamicin. Inanother embodiment, the invention encompasses a compound of Formula Vwherein the organic molecule is benzonitrile, 2-cyanophenol or3-butenenitrile.

In an embodiment, the present invention encompasses a compound ofFormula VI, wherein the organic molecule is 4-AP. In another embodiment,the invention encompasses a compound of Formula VI, wherein the organicmolecule is TzGly. In another embodiment, the invention encompasses acompound of Formula VI, wherein the organic molecule is (tetrazol-5-yl)AMPA. In another embodiment, the invention encompasses a compound ofFormula VI, wherein the organic molecule is nicotine or caffeine. Inanother embodiment, the invention encompasses a compound of Formula VI,wherein the organic molecule is serotonin, epinephrine, norepinephrine,or dopamine. In another embodiment, the present invention encompasses acompound of Formula VI, wherein the organic molecule is adenosine5′-diphosphate ADP, adenosine 5′-triphosphate ATP, adenosine5′-monophosphate AMP, cyclic adenosine 5′-diphosphate ribose, oradenosine 3′,5′-cyclicmonophosphate. In another embodiment, theinvention encompasses a compound of Formula VI wherein the organicmolecule is aminobutyric acid or L-glutamic acid, or methyl-D-asparticacid. In another embodiment, the invention encompasses a compound ofFormula VI wherein the organic molecule is methionine,methylthiogalactose or isopropylthiogalactose. In another embodiment,the invention encompasses a compound of Formula VI wherein the organicmolecule is methylthiogalactose or isopropylthiogalactose. In anotherembodiment, the invention encompasses a compound of Formula VI whereinthe organic molecule is mercaptopurine, thioguanine, doxorubicin,cytarabin, temozolomide, or gentamicin. In another embodiment, theinvention encompasses a compound of Formula VI wherein the organicmolecule is mercaptopurine. In another embodiment, the inventionencompasses a compound of Formula VI wherein the organic molecule isthioguanine. In another embodiment, the invention encompasses a compoundof Formula VI wherein the organic molecule is doxorubicin. In anotherembodiment, the invention encompasses a compound of Formula VI whereinthe organic molecule is cytarabin. In another embodiment, the inventionencompasses a compound of Formula VI wherein the organic molecule istemozolomide. In another embodiment, the invention encompasses acompound of Formula VI wherein the organic molecule is gentamicin. Inanother embodiment, the invention encompasses a compound of Formula VIwherein the organic molecule is benzonitrile, 2-cyanophenol or3-butenenitrile.

In an embodiment, the present invention encompasses a compound ofFormula VII, wherein the organic molecule is 4-AP. In anotherembodiment, the invention encompasses a compound of Formula VII, whereinthe organic molecule is TzGly. In another embodiment, the inventionencompasses a compound of Formula VII, wherein the organic molecule is(tetrazol-5-yl) AMPA. In another embodiment, the invention encompasses acompound of Formula VII, wherein the organic molecule is nicotine orcaffeine. In another embodiment, the invention encompasses a compound ofFormula VII, wherein the organic molecule is serotonin, epinephrine,norepinephrine, or dopamine. In another embodiment, the presentinvention encompasses a compound of Formula VII, wherein the organicmolecule is adenosine 5′-diphosphate ADP, adenosine 5′-triphosphate ATP,adenosine 5′-monophosphate AMP, cyclic adenosine 5′-diphosphate ribose,or adenosine 3′,5′-cyclicmonophosphate. In another embodiment, theinvention encompasses a compound of Formula VII wherein the organicmolecule is aminobutyric acid or L-glutamic acid, or methyl-D-asparticacid. In another embodiment, the invention encompasses a compound ofFormula VII wherein the organic molecule is methionine,methylthiogalactose or isopropylthiogalactose. In another embodiment,the invention encompasses a compound of Formula VII wherein the organicmolecule is methylthiogalactose or isopropylthiogalactose. In anotherembodiment, the invention encompasses a compound of Formula VII whereinthe organic molecule is mercaptopurine, thioguanine, doxorubicin,cytarabin, temozolomide, or gentamicin. In another embodiment, theinvention encompasses a compound of Formula VII wherein the organicmolecule is mercaptopurine. In another embodiment, the inventionencompasses a compound of Formula VII wherein the organic molecule isthioguanine. In another embodiment, the invention encompasses a compoundof Formula VII wherein the organic molecule is doxorubicin. In anotherembodiment, the invention encompasses a compound of Formula VII whereinthe organic molecule is cytarabin. In another embodiment, the inventionencompasses a compound of Formula VII wherein the organic molecule istemozolomide. In another embodiment, the invention encompasses acompound of Formula VII wherein the organic molecule is gentamicin. Inanother embodiment, the invention encompasses a compound of Formula VIIwherein the organic molecule is benzonitrile, 2-cyanophenol or3-butenenitrile.

In an embodiment, the present invention encompasses a compound ofFormulas I-III and V-VIII, wherein the labeling molecule is rhodamine.In another embodiment, the invention encompasses a compound of FormulasI-III and V-VIII, wherein the labeling molecule is fluorescein. Inanother embodiment, the invention encompasses a compound of FormulasI-III and V-VIII, wherein the labeling molecule is iodeosin. In anotherembodiment, the invention encompasses a compound of Formulas I-III andV-VIII, wherein the labeling molecule is a fluorescent moleculecontaining a —CN group whose nitrogen atom forms a bond with Ru. Inanother embodiment, the invention encompasses a compound of FormulasI-III and V-VIII, wherein the labeling molecule is a fluorescentmolecule containing a pyridyl group whose nitrogen atom forms a bondwith Ru. In another embodiment, the invention encompasses a compound ofFormulas I-III and V-VIII, wherein the labeling molecule is afluorescent molecule containing an amino group whose nitrogen atom formsa bond with Ru. In another embodiment, the invention encompasses acompound of Formulas I-III and V-VIII, wherein the labeling molecule isa fluorescent molecule containing a phosphine group whose nitrogen atomforms a bond with Ru.

In another embodiment, the invention encompasses a compound of FormulasI-III and V-VIII, wherein the labeling molecule is rhodamine containinga —CN group whose nitrogen atom forms a bond with Ru. In anotherembodiment, the invention encompasses a compound of Formulas I-III andV-VIII, wherein the labeling molecule is rhodamine containing a pyridylgroup whose nitrogen atom forms a bond with Ru. In another embodiment,the invention encompasses a compound of Formulas I-III and V-VIII,wherein the labeling molecule is rhodamine containing an amino groupwhose nitrogen atom forms a bond with Ru. In another embodiment, theinvention encompasses a compound of Formulas I-III and V-VIII, whereinthe labeling molecule is rhodamine containing a phosphine group whosenitrogen atom forms a bond with Ru.

In some embodiments, of the compounds of Formula I-III and V-VIII, L²is:

(RhodB-MAMePy) or a chloride salt thereof (RhodB-MAMePy-Cl).

In some embodiments of the compounds of Formula I-III and V-VIII, L² is:

(Rhod6G-MAMePy) or a chloride salt thereof (Rhod6G-MAMePy-Cl).

In some embodiments, the labeling molecule or an active derivativethereof is bonded to Ru to Ru through other coordinating groups: —NH₂,pyridyl, or a phosphine group.

In some embodiments of Photolabile Compounds, L² comprises a labelingmolecule or an active derivative thereof, which can serve as a lightantenna and can transfer energy to the portion of the compound thateffects photorelease of moiety L¹. This way the light gathering becomesdissociated from the chemical release step, enabling orthogonal chemicalstrategies for both processes. Thus, in some embodiments, a PhotolabileCompound has a dual functionalization, one being a light antenna andsecond being a photorelease entity.

In one aspect, a Photolabile Compound can be used in combinatorialchemistry strategies. For example, one could generate a systematicseries of derivatives in the photorelease part of the molecule andperform large-scale screening with them. Alternatively or in addition,one could fix the photorelease part of the molecule while derivatizingthe light antenna moiety.

In another aspect, the magnetic and/or radio-opaque properties of aPhotolabile Compound are used as a means of identifying it ormanipulating it in living cells. In one such embodiment, PhotolabileCompounds are used as MRI (Magnetic Resonance Imaging) contrast agents.

When L² comprises a labeling molecule such as a fluorophore or aderivative thereof, L² can servean antenna, to extend the irradiationspectrum to lower energies with very high absorption and efficiency. Inspecific embodiments, the fluorescent dye is linked through aderivatization using a non-releasable ligand, i.e., a phosphine(:P(R)₂-fluorophore). In these embodiments, the light is collected bythe fluorescent moiety acting as an antenna, and transferred to the Rucenter, populating the MLCT or d-d excited states. Once the excitedstate is reached, the other ligand (i.e., L¹, which can be a biomoleculeor drug) is released.

The fact that in this case the active wavelength is not determined bythe Ru center but by the high absorption of the fluorescent dye(˜100,000 M⁻¹ cm⁻¹) allows the tuning of different complexes havingdifferent photouncageable drugs at different wavelengths, from 400 to600 nm, providing orthogonality.

Use of a suitable organic dye coordinated to the ruthenium next to thecoordinated fluorescent dye (i.e., a labeling molecule) permits theselective quenching of the fluorescence due to its proximity. In thiscase, the absorption of light via the Ru-bpy MLCT band (without antennaeffect, the fluorescent being quenched by the nearby nonfluorescentdye), can photorelease either the fluorescent dye, increasingdramatically the fluorescence and “uncaging” the fluorophore, or thenonfluorescent quencher, with somewhat similar results (under someconditions, however, the presence of the metallic center can quench thefluorophore).

Further in accordance with the invention, the compounds of FormulasV-VIII, wherein R¹ and R² and/or R³ and R⁴ combine to form a carbocyclicring substituted by one or more oxo groups, can attach to surfaces andare useful for the delivery of compounds to surfaces. For example, suchcompounds can bind to nanoparticles or nanopowders and be used todeliver biologically active organic molecules or labeling molecules.Nanoscale delivery systems have substantial applications in thecontrolled and targeted transport of drugs. Drugs can be carriedselectively to targeted cells by means of nanoparticles with specificsurface fictionalization. Nanoparticles can penetrate cell membranes andovercome physiological barriers in the organism, and nanoparticles canalso improve the solubility and bio-availability of drugs.

In another embodiment, the invention provides a method for enhancing thesolubility of an organic molecule, comprising complexing an organicmolecule to a photolabile caging group to form a Photolabile Compound,such that exposing the compound to light under sufficient conditionsreleases the organic molecule from the compound. In an embodiment, theorganic molecule has:

(i) a 5-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with Ru;

(ii) a 6-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with Ru;

(iii) an 8-10-membered bicyclic ring, one of the bicyclic rings beingaromatic and having a nitrogen atom member that forms a bond with Ru;

(iv) an —NH₂ group whose nitrogen atom forms a bond with Ru;

(v) a —COOH group, one of whose oxygen atoms forms a bond with Ru;

(vi) a —PR₂ group whose phosphorus atom forms a bond with Ru, wherein Ris independently —H, —C₁-C₁₈ alkyl, or aryl;

(vii) an —SR group whose sulfur atom forms a bond with Ru, wherein R isindependently —H, —C₁-C₁₈ alkyl, or aryl; or

(viii) a —CN group whose nitrogen atom forms a bond with Ru.

In another embodiment, the organic molecule has:

(i) a 5-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with Ru;

(ii) a 6-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with Ru;

(iii) an 8-10-membered bicyclic ring, one of the bicyclic rings beingaromatic and having a nitrogen atom member that forms a bond with Ru;

(iv) an —NH₂ group whose nitrogen atom forms a bond with Ru;

(v) a —COOH group, one of whose oxygen atoms forms a bond with Ru;

(vi) a —PR₂ group whose phosphorus atom forms a bond with Ru, wherein Ris independently —H, —C₁-C₁₈ alkyl, or aryl; or

(vii) an —SR group whose sulfur atom forms a bond with Ru, wherein R isindependently —H, —C₁-C₁₈ alkyl, or aryl.

In an embodiment, the present invention encompasses a compositioncomprising an effective amount of a Photolabile Compound and aphysiologically acceptable carrier, vehicle, diluent, or excipient.Suitable carriers, vehicles, diluents, or excipients are known to thoseskilled in the art and include, without limitation, physiologicallysterile saline and others as described herein.

In another embodiment, the present invention provides a vesselcontaining a Photolabile Compound. The vessel can further contain abiological sample, wherein the sample is, for example, hair, an organspecimen; a tissue or cell, for example, a neuronal tissue or cell; atumor or cancer or neoplastic tissue or cell; or a tissue or cellremoved from a patient or subject of interest. Tissue specimens slicedfrom microtomes, for example, are examples of suitable biologicalsamples.

Any type of vessel that is capable of transmitting the wavelengths oflight used for releasing the organic molecules or labeling moleculescomprising the Photolabile Compounds, and that is inert to solvent inwhich a Photolabile Compound is suspended, is suitable for use. Forexample, the vessel can be made of glass, plastic, acrylic, quartz, anoble metal, etc. In addition, if the vessel is composed of, or encasedin, metal, e.g., aluminum, titanium, or stainless steel, exposure tolight is performed through the top of the vessel, or through a “window”or other light-penetrable opening in the vessel. For solid-likematerials, acrylic plastic or acrylamide-bisacrylamide gel, etc., forexample, can be used as media in which the Photolabile Compounds arecontained. For example, an acrylic plastic coating formulated using aCHCl₃ solution of acrylic and a Ru(bpy) complex changed its spectrumfollowing irradiation, thus allowing photorelease in a solid state. Forsuch solid state aspects of the invention, the temperature may be keptat 4K.

Solvents suitable in which a Photolabile Compounds can be exposed tolight include aqueous solvents; water; acetonitrile; alcohol, e.g.,methanol, ethanol; acetone; chlorinated solvents such as CH₂Cl₂ andCHCl₃; or dimethylsulfoxide.

Suitable temperatures at which a Photolabile Compound is exposed tolight range, in general, from about 0° C. to about 100-150° C.

In another embodiment, this invention encompasses a method for releasingan organic molecule or a labeling molecule from a Photolabile Compound.The method comprises exposing a Photolabile Compound to light underconditions sufficient to release the organic molecule or labelingmolecule from the compound. In the method, the light comprises awavelength of about 300 to about 500 nm, or about 300 to about 360 nm,or about 450 to about 500 nm. Further, the exposing can occur at atemperature from about 0° C. to about 150° C. In an embodiment, themethods of the invention comprise a Photolabile Compound, e.g., acompound of Formula I-VIII, light of a wavelength of about 300 nm toabout 500 nm; L¹ being L², and a temperature of about 0° C. to about150° C. In another embodiment, the methods comprise a PhotolabileCompound, light of a wavelength of about 300 nm to about 360 nm; L¹being L², and a temperature of about 0° C. to about 150° C. In anotherembodiment, the methods comprise a Photolabile Compound, light of awavelength of about 450 nm to about 500 nm; L¹ being L², and atemperature of about 0° C. to about 150° C. In another embodiment, themethods comprise a Photolabile Compound, visible or infrared light; L¹being L², and a temperature of about 0° C. to about 150° C.

In an embodiment, the invention encompasses a method for assaying anorganic molecule, comprising exposing a Photolabile Compound and abiological sample to light under conditions sufficient to release theorganic molecule from the Photolabile Compound, and (b) determining aneffect of the organic molecule on the biological sample. The sample canbe a biological sample, such as a sample excised, removed, or otherwisetaken from a subject's body. The subject's biological sample can be, forexample, a hair sample, an organ or tissue sample, e.g., from a biopsyor an autopsy, or a cell sample. In addition, the biological sample canbe a body fluid sample. Body fluid samples include, without limitation,blood, serum, plasma, lymph, saliva, sputum, tears, semen, or urine.Biological samples can further include, without limitation, braintissue, brain cells, muscle tissue, muscle cells, muscle fibers,fibroblasts, tissue slices, or fine tissue specimens, from any organ ofthe body, sarcoplasmic reticulum, skin tissue, membrane preparations orfragments, etc.

The light for exposing the compounds according to the methods of thisinvention can be sunlight, photo-optic light, or laser light.Advantageously, in the methods of this invention, the light for exposingthe compound is other than UV radiation. Thus, for example, the lightcan be visible light or infrared light, including one-photon andtwo-photon light. The light can be emitted from a variety of sources,including without limitation, a laser light source, a tungsten lightsource, a photooptic light source, etc. Another advantage of visiblelight to expose or irradiate the compounds of the invention relates tothe convenience and ability to use a visible light microscope, forexample, to view a sample into which a compound is introduced and tomicroscopically visualize or monitor a photoreleased ligand from thecompound after exposure to visible light. Because many microscopes donot transmit UV light, it is advantageous to be able to use a non-quartzmicroscope in accordance with this invention. Yet another advantage tothe use of visible light is that it is not detrimental to living cellsand tissues, making it beneficial for in vivo patient use. In addition,for patient use, the light can be specifically directed to an area wherea Photolabile Compound is introduced or administered by the use of lasertechnology, fibers, probes, tubes, and the like. Such probes, fibers, ortubes can be directly inserted, for example, into a body cavity oropening, or under or through the skin, to expose the PhotolabileCompound to light.

In another of its embodiments, the present invention includes a methodof making an organic molecule bioavailable to a subject. The organicmolecule can be made bioavailable to a localized body region or area ofthe subject, or systemically to the whole body. Local bioavailability ofthe Photolabile Compounds is achieved, for example, via delivery devicesand methods that allow the compounds to be directly administered, forexample, inserted into a body cavity, or opening, or through or into theskin. The method of this embodiment involves administering a PhotolabileCompound to the subject, and exposing the compound to light underconditions sufficient to release the organic molecule from the compound,thereby making the organic molecule bioavailable to the subject, and/orto a body site or region of the subject. The exposure to light cancomprise the use of probes, fibers, tubes, and the like, which allow thelight to be specifically directed to the area of interest on or withinthe body. Alternatively, the Photolabile Compounds can be administeredto the patient kept in the dark; for photorelease of the organicmolecule, the patient can be moved to the light where exposure to lightand photorelease occur. In an embodiment according to this method, theorganic molecule has:

(a) a 5-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with Ru;

(b) a 6-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with Ru;

(c) an 8-10-membered bicyclic ring, one of the bicyclic rings beingaromatic and having a nitrogen atom member that forms a bond with Ru;

(d) an —NH₂ group whose nitrogen atom forms a bond with Ru;

(e) a —COOH group, one of whose oxygen atoms forms a bond with Ru;

(f) a —PR₂ group whose phosphorus atom forms a bond with Ru, wherein Ris independently —H, —C₁-C₁₈ alkyl, or aryl;

(g) an —SR group whose sulfur atom forms a bond with Ru, wherein R isindependently —H, —C₁-C₁₈ alkyl, or aryl; or

(h) a —CN group whose nitrogen atom forms a bond with Ru.

In another embodiment according to this method, the organic moleculehas:

(a) a 5-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with Ru;

(b) a 6-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with Ru;

(c) an 8-10-membered bicyclic ring, one of the bicyclic rings beingaromatic and having a nitrogen atom member that forms a bond with Ru;

(d) an —NH₂ group whose nitrogen atom forms a bond with Ru;

(e) a —COOH group, one of whose oxygen atoms forms a bond with Ru;

(f) a —PR₂ group whose phosphorus atom forms a bond with Ru, wherein Ris independently —H, —C₁-C₁₈ alkyl, or aryl; or

(g) an —SR group whose sulfur atom forms a bond with Ru, wherein R isindependently —H, —C₁-C₁₈ alkyl, or aryl.

In another embodiment, the organic molecule has:

(a) a tetrazolyl group, one of its nitrogen atoms forming a bond withRu;

(b) nicotine or caffeine, whose pyridyl nitrogen atom forms a bond withRu;

(c) an 8-10-membered bicyclic ring, one of the bicyclic rings beingaromatic and having a nitrogen atom member that forms a bond with Ru;

(d) an —NH₂ group whose nitrogen atom forms a bond with Ru;

(e) a —COOH group, one of whose oxygen atoms forms a bond with Ru;

(f) a —PR₂ group whose phosphorus atom forms a bond with Ru, wherein Ris independently —H, —C₁-C₁₈ alkyl, or aryl;

(g) an —SR group whose sulfur atom forms a bond with Ru, wherein R isindependently —H, —C₁-C₁₈ alkyl, or aryl; or

(h) a —CN group whose nitrogen atom forms a bond with Ru.

In another embodiment, the organic molecule has:

(a) a tetrazolyl group, one of its nitrogen atoms forming a bond withRu;

(b) nicotine or caffeine, whose pyridyl nitrogen atom forms a bond withRu;

(c) an 8-10-membered bicyclic ring, one of the bicyclic rings beingaromatic and having a nitrogen atom member that forms a bond with Ru;

(d) an —NH₂ group whose nitrogen atom forms a bond with Ru;

(e) a —COOH group, one of whose oxygen atoms forms a bond with Ru;

(f) a —PR₂ group whose phosphorus atom forms a bond with Ru, wherein Ris independently —H, —C₁-C₁₈ alkyl, or aryl; or

(g) an —SR group whose sulfur atom forms a bond with Ru, wherein R isindependently —H, —C₁-C₁₈ alkyl, or aryl.

In a related embodiment, the Photolabile Compounds are useful forreleasing an organic molecule, such as a drug, pharmaceutical, smallbiologically active molecule, and the like as described above. Releaseof the organic molecule from the Photolabile Compound allows the organicmolecule to become bioavailable to a subject, or patient, afflicted witha disease, disorder, pathology, or condition. The Photolabile Compoundsand organic molecules are useful in veterinary and human medicine.Diseases, disorders, pathologies, or conditions for which making anorganic molecule bioavailable would serve to treat, ameliorate, reduce,eliminate, abate, or prevent the disease, disorder, pathology, orcondition are further described below and include, as nonlimitingexamples, peripheral and central nervous system disorders, neurologicaldisorders and disorders related thereto, neurodegenerative disorders anddisorders related thereto, epilepsy, seizures, migraines, headaches,stroke, anxiety, depression, restricted brain function, addictivedisorders, neuroses, psychoses, pruritic conditions, Parkinson'sdisease, Huntington's chorea, cognitive disorders, memory lapses,Alzheimer's disease, dementia, dyskinesia, muscle spasms, retinopathy,vomiting, cancers, neoplasms, tumors, vascular diseases, andcardiovascular diseases.

As further, yet non-limiting examples, the organic molecule is aneurochemical that blocks potassium channels for use, for example, intreating neurodegenerative, or neurological diseases or disorders. In aparticular embodiment, the organic molecule is 4-AP, which is a calciumchannel blocker. In another embodiment, the organic molecule is TzGly,which is an NMDA-receptor agonist that is more potent than NMDA. In oneembodiment, for making an organic molecule of the invention bioavailableto a subject in need thereof, the exposure of the Photolabile Compoundto light can occur at the site of the disease, disorder, pathology, orcondition, such as a site of a tumor, neoplasm, or cancer lesion orgrowth, thereby releasing the organic molecule locally and moreprecisely at the needed location. In another embodiment, for making anorganic molecule of the invention bioavailable to a subject in needthereof, the exposure of the Photolabile Compound to light can occur atthe sight of a blood dyscrasia.

In other related embodiments, the present invention provides methods fortreatment, therapy, and prophylaxis by administering an effective amountof a Photolabile Compound, or a physiologically acceptable compositioncomprising a Photolabile Compound to a subject, so as to make an organicmolecule bioavailable to the subject. The Photolabile Compound can besubstantially purified (e.g., substantially free from substances thatlimit its effect or produce undesired side-effects). Advantageously, formethods in which a Photolabile Compound is administered to a subject,the light, e.g., infrared, laser, or visible light, for photoreleasingthe organic molecule to make it bioavailable to the subject can bedirected to an internal site or region of interest by using photoopticdevices, probes and fibers, such as are known in the art and describedsupra. Those having skill in the art can employ, manipulate, andinternally direct the devices for exposing a Photolabile Compound tolight after the Photolabile Compound is administered to a subject.

In the methods of the present invention involving subjects, and/or thetreatment, therapy, or prophylaxis of a disease, disorder, pathology, orcondition, the subject is preferably an animal, including but notlimited to, mammals such as human and non-human primates, cows, pigs,horses, goats, sheep, rabbits, chickens, cats, dogs, guinea pigs, rats,mice, etc. The methods of the invention especially encompass humantreatments.

Various delivery systems are known and can be used to administer aPhotolabile Compound, e.g., in sterile solution, encapsulation inliposomes, microparticles, microcapsules, or receptor-mediatedendocytosis (See, e.g., Wu and Wu, 1987, J. Biol. Chem., 262:4429-4432).Methods of introduction include, but are not limited to, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, topical, transdermal, parenteral, intrathecal, vaginal,rectal, colorectal, oral, intracranial, retroorbital, intrasternalroutes, or a combination thereof.

The Photolabile Compounds or compositions may be administered by anyconvenient route or mode, for example, by continuous infusion,non-continuous infusion, or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, epidermis,rectal and intestinal mucosa, etc.) and may be administered togetherwith other biologically active and/or therapeutic agents. Administrationcan be systemic or local. In addition, it may be desirable to introducethe Photolabile Compounds or compositions into the central nervoussystem by any suitable route, including intraventricular and intrathecalinjection; intraventricular injection may be facilitated by anintraventricular catheter, for example, attached to a reservoir, such asan Ommaya reservoir. Pulmonary administration can also be employed,e.g., by use of an inhaler or nebulizer, and formulation with anaerosolizing agent.

In a particular embodiment, it may be desirable to administer thePhotolabile Compounds or compositions locally to the area in need oftreatment. This may be achieved, for example, by local infusion duringsurgery, topical application, e.g., in conjunction with a wound dressingafter surgery, by injection, by means of a catheter, by means of asuppository, or by means of an implant, where the implant is a porous,non-porous, or gelatinous material, including membranes, such assialastic membranes, or fibers.

In another embodiment involving topical administration, a transdermalpatch can be used. In accordance with this embodiment, the PhotolabileCompound remains unexposed to light until the patch is manipulated by apatient or medical provider so that all or a portion of the patchcontaining a Photolabile Compound is exposed to light. Accordingly, thepatch can be opened and the bioactive molecule released, or “activated”from the compound after exposure to light, for example, by the patient'smoving from a dark room to a lighted room, or from a dark area to alight area; by the patient's directly exposing the patch, or a portionthereof, to a suitable light source, or by the patient's exposing all ora portion of the patch to daylight. A variety of types of transdermalpatches are known and used by the skilled practitioner in the art.Alternatively for topical administration, a Photolabile Compound can beformulated into a light-sensitive composition, which is contained in adark, light-protected container, and applied topically to the area ofinterest, e.g., applied to or rubbed onto the skin of a subject, in thedark. Following topical application in the dark, the area of interest isexposed to light, or to an appropriate light source, or the subjectmoves into the light, thereby causing the organic molecule of thePhotolabile Compound to be released.

In another embodiment, the Photolabile Compounds or compositions can bedelivered in a vesicle, in particular a liposome (See, e.g., Langer,1990, Science, 249:1527-1533; Treat et al., In: Liposomes in the Therapyof Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.),Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327;see generally ibid.) In yet another embodiment, the PhotolabileCompounds or compositions can be delivered in a controlled-releasesystem. For example, a pump may be used (see Langer, supra; Sefton,1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980,Surgery, 88:507; Saudek et al., 1989, NEJM, Med. 321:574 (1989)), orpolymeric materials can be used (See, e.g., Medical Applications ofControlled Release, Langer and Wise (eds.), CRC Press, Boca Raton, Fla.(1974); Controlled Drug Bioavailability, Drug Product Design andPerformance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger andPeppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem., 23:61; Levy etal., 1985, Science, 228:190; During et al., 1989, Neurol., 25:351; andHoward et al., 1989, J. Neurosurg., 71:105). Moreover, acontrolled-release system can be placed proximal to the therapeutictarget, e.g., the brain, thus requiring only a fraction of the systemicdose (see, e.g., Goodson, 1984, In: Medical Applications of ControlledRelease, Vol. 2, pp. 115-138). As further guidance, other controlledrelease systems are found in Langer, 1990, Science, 249:1527-1533.

The Photolabile Compounds are also provided in effective amounts inpharmaceutical compositions comprising a pharmaceutically acceptablecarrier, diluent, excipient, or vehicle, for example, for use astherapeutics. In one embodiment, the term “pharmaceutically acceptable”refers to approval by a regulatory agency of the Federal or a stategovernment, or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly, inhumans. The terms vehicle, carrier, or excipient refer to a diluent oradjuvant in or with which the therapeutic is administered. Suchpharmaceutical carriers, vehicles, or excipients can be sterile liquids,such as water and oils, including those of petroleum, animal, vegetableor synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when a pharmaceuticalcomposition is administered intravenously and is water soluble. Salinesolutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. If needed or desired,the composition of the invention can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents.

The Photolabile Compounds and compositions of the present invention canbe formulated as solutions, suspensions, emulsions, tablets, pills,capsules, powders, sustained-release formulations and the like. Thecompositions can be formulated as a suppository, with traditionalbinders and carriers such as triglycerides. Oral formulations caninclude standard carriers, such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Examples of suitable pharmaceutical carriersand the like are described in the current edition of “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions routinelycontain a therapeutically effective amount of the Photolabile Compound,preferably in purified form, together with a suitable amount of carrierso as to provide the form for proper administration to a subject. Theformulation should suit the mode of administration.

In another embodiment, a Photolabile Compound of the invention isformulated in accordance with routine procedures as a pharmaceuticalcomposition adapted for intravenous administration to human beings.Typically, compositions for intravenous administration are solutions insterile isotonic aqueous buffer. Where necessary, the composition mayalso include a solubilizing agent and a local anesthetic such aslidocaine to ease pain at the site of the injection. Generally, theingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water freeconcentrate in a hermetically sealed container, such as an ampoule orsachette indicating the quantity of active agent. Where the compositionis to be administered by infusion, it can be dispensed with an infusionbottle containing sterile pharmaceutical grade water or saline. Wherethe composition is administered by injection, an ampoule of sterilewater for injection or saline can be provided so that the ingredientsmay be mixed prior to administration. Sterility for of a composition fortherapeutic administration is readily accomplished by filtration throughsterile filtration membranes (e.g., 0.2 micron membranes). Therapeuticsgenerally are placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having a stopper pierceableby a hypodermic injection needle. Therapeutics are typically stored inunit or multi-dose containers, for example, sealed ampoules or vials, asan aqueous solution or as a lyophilized formulation for reconstitution.Where necessary, the ampoule or vial is essentially impenetrable bylight. As an example of a lyophilized therapeutic formulation, 10-m1vials are filled with 5 ml of sterile-filtered 1% (w/v) aqueoustherapeutic solution, and the resulting mixture is lyophilized. Theinfusion solution is prepared by reconstituting the lyophilizedtherapeutic using bacteriostatic Water-for-Injection.

The amount of a Photolabile Compound which will be effective in thetreatment, amelioration, reduction, elimination, inhibition, orprevention of a particular disease, condition, pathology, or disorderassociated with the use and bioactivity of an organic molecule can bedetermined by standard clinical techniques. An “effective amount” or a“pharmaceutically effective amount” of a Photolabile Compound of thisinvention refers to an amount effective for treating, ameliorating,reducing, abating, eliminating, preventing, a disease, condition,pathology, or disorder for which the compound is being used. Inparticular embodiments, an effective amount is an amount effective formaking an organic molecule of the invention bioavailable to a subject.If another therapeutic agent is used in conjunction with the PhotolabileCompounds, the effective amount of the therapeutic agent refers to anamount effective for providing the therapeutic effect of the therapeuticagent. The precise dose to be employed in the formulation will alsodepend on the route of administration, as well as an individualpatient's circumstances, such as age, health and vital statistics of thepatient, and the severity of the disease, condition, or disorder. Dosingshould be decided according to the judgment of the medical practitionerbased on an evaluation of the patient and considerations of a patient'sphysiologic situation and medical history. In addition, in vitro assaysmay optionally be used to assist in determining optimal dosage ranges.Effective doses can be extrapolated from dose-response curves derivedfrom in vitro or in vivo animal model test systems.

As general guidance, the total effective amount of a PhotolabileCompound administered parenterally per dose will be in the range ofabout 1 μg/kg/day to 10 mg/kg/day of a subject's body weight, although,as noted above, this will be subject to discretion based on thesubject's condition and the above-mentioned variables. A therapeuticdose can also be at least 0.01 mg/kg/day, or between about 0.01 and 1mg/kg/day, with particular regard for human administration. If givencontinuously, a therapeutic is typically administered at a dose rate ofabout 1 μg/kg/hour to about 50 μg/kg/hour, either by 1 to 4 injectionsper day or by continuous subcutaneous infusions, e.g., using amini-pump. An intravenous bag solution may also be employed. The lengthof treatment needed to observe changes and the interval followingtreatment for responses to occur will likely vary depending on thedesired effect. In some embodiments, suitable effective dosage amountsrange from about 10 μg to about 2500 mg about every 4 hours, althoughthe amounts are typically about 100 mg or less. In one embodiment, theeffective dosage of a Photolabile Compound ranges from about 0.01 mg toabout 100 mg about every 4 hours. In another embodiment, the effectivedosage of a compound of the invention ranges from about 0.020 mg toabout 50 mg every 4 hours, and in another embodiment, about 0.025 mg toabout 20 mg about every 4 hours. The effective dosage amounts refer tototal amounts administered. Thus, if more than one of the PhotolabileCompounds is administered, the effective dosage amounts correspond tothe total amount administered.

In another embodiment, if a Photolabile Compound is contacted with abiological sample in vitro, an effective amount will typically rangefrom about 0.01 μg/L to about 5 mg/L; in another embodiment from about0.01 μg/L to about 2.5 mg/L; in another embodiment, from about 0.01 μg/Lto about 0.5 mg/L; and in yet another embodiment, from about 0.01 μg/Lto about 0.25 mg/L of a solution or suspension of a pharmaceuticallyacceptable carrier, diluent, or excipient. In an embodiment, the volumeof solution or suspension is from about 1 μL to about 1 mL; in anotherembodiment, the volume of solution or suspension is about 200 μL.

Examples of neoplastic or hyperproliferative diseases, disorders,pathologies, or conditions that can be treated, ameliorated, reduced,abated, eliminated, inhibited, prevented, and/or diagnosed using thePhotolabile Compounds and photoreleased organic molecules include, butare not limited to, neoplasms (cancers or tumors) located in the colon,abdomen, bone, breast, digestive system, esophagus, liver, pancreas,peritoneum, endocrine glands (adrenal, parathyroid, pituitary,testicles, ovaries, cervix, thymus, thyroid), eye, head and neck,nervous (central and peripheral), lymphatic system, pelvis, skin, softtissue, spleen, thoracic areas, bladder, and urogenital system. Cancersthat may be treatable using the Photolabile Compounds include follicularlymphomas, carcinomas with p53 mutations, and hormone-dependent tumors,including, but not limited to colon cancer, cardiac tumors, pancreaticcancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinalcancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma,lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi'ssarcoma and ovarian cancer, or metastases thereof. Autoimmune diseases,disorders, or conditions may be treatable with the Photolabile Compoundsand include multiple sclerosis, Sjogren's syndrome, Hashimoto'sthyroiditis, biliary cirrhosis, Bechet's disease, Crohn's disease,polymyositis, systemic lupus erythematosus and immune-relatedglomerulonephritis, rheumatoid arthritis, ischemic injury (e.g., causedby myocardial infarction, stroke and reperfusion injury), liver injury(e.g., hepatitis related liver injury, ischemia/reperfusion injury,cholestosis (bile duct injury) and liver cancer); toxin-induced liverdisease (e.g., caused by alcohol), septic shock, cachexia and anorexia.Viral infections (such as herpes viruses, pox viruses and adenoviruses),inflammation, graft versus host (GVH) disease, acute graft rejection,and chronic graft rejection may also be treatable with the PhotolabileCompounds.

Additional diseases or conditions associated with abnormal and increasedcell survival that may be treated, ameliorated, reduced, abated,eliminated, inhibited, prevented, and/or diagnosed using the PhotolabileCompounds include, but are not limited to, progression and/or metastasesof malignancies and related disorders such as leukemia (including acuteleukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia,including myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia) and chronic leukemias (e.g., chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma, and retinoblastoma.

In another embodiment, the Photolabile Compounds may be needed astherapeutics to stimulate epithelial cell proliferation and basalkeratinocytes for the purpose of wound healing, and to stimulate hairfollicle production and the healing of dermal wounds. The PhotolabileCompounds of the invention may be clinically useful in stimulating woundhealing, including surgical wounds, excisional wounds, deep woundsinvolving damage of the dermis and epidermis, eye tissue wounds, dentaltissue wounds, oral cavity wounds, diabetic ulcers, dermal ulcers,cubitus ulcers, arterial ulcers, venous stasis ulcers, burns resultingfrom heat exposure or chemicals, and other abnormal wound healingconditions such as uremia, malnutrition, vitamin deficiencies andcomplications associated with systemic treatment using steroids,radiation therapy, anti-neoplastic drugs and anti-metabolites.

Other diseases, disorders, or conditions that may be treated,ameliorated, reduced, abated, eliminated, inhibited, prevented, and/ordiagnosed with the Photolabile Compounds include AIDS; neurodegenerativediseases, disorders, and/or conditions (such as Alzheimer's disease,Parkinson's disease, amyotrophic lateral sclerosis (ALS), multiplesclerosis, Retinitis pigmentosa (RP), cerebellar degeneration and braintumor or prior associated disease).

In one embodiment, diseases and conditions that are treatable usingcalcium channel blockers, e.g., 4AP, include without limitation, heartdisease, hypertension, angina, chest pain, cardiovascular diseases, suchas coronary artery disease, cardiomyopathies, valvular heart disease,renal disease, Peyronie's disease and neurological, neurophysiological,or neuromuscular diseases and conditions, e.g., amyotrophic lateralsclerosis (ALS), multiple sclerosis, and epilepsy.

In another embodiment, diseases that are treatable using NMDA receptoragonists or antagonists, e.g., TzGly, include without limitation,neurological, neurodegenerative, or neurophysiological diseases,disorders, and conditions, such as Alzheimer's disease, Parkinson'sdisease, Huntington's disease, and dyskinesias. In another embodiment,neurological, neurodegenerative, and neurophysiological diseases, e.g.,Parkinson's disease, Alzheimer's disease, etc., are treatable usingTz-AMPA.

In another embodiment, the present invention relates to kits comprisinga Photolabile Compound and instructions for use. A kit may be used in adiagnostic, screening, or testing assay. A kit may also be apharmaceutical pack, particularly for use in treating or preventing adisease, disorder, pathology, or condition. A kit for pharmaceutical useis typically sterile and contains a Photolabile Compound in an amounteffective to treat or prevent a disease, disorder, pathology, orcondition, and a pharmaceutically acceptable carrier, diluent, orexcipient. The kit, or a pharmaceutical pack, can comprise one or morevessels or containers filled with an effective amount, e.g., unit dosageform, of one or more of the Photolabile Compounds or compositions of theinvention, and a pharmaceutically acceptable carrier, diluent, orexcipient. The kit, or pharmaceutical pack, can further comprise alabel. In addition, the kit, or pharmaceutical pack, can also include aunit dosage form of another therapeutic agent, for example, a containercontaining an effective amount of the other therapeutic agent. The kit,or pharmaceutical pack, may further optionally contain a notice in theform prescribed by a governmental agency regulating the manufacture,use, or sale of pharmaceuticals or biological products, reflectingapproval by the agency of manufacture, use or sale for humanadministration. The kit, or pharmaceutical pack, can also contain adevice useful for administering the unit dosage forms. Examples of suchdevices include, without limitation, a syringe, a drip bag, a patch, aninhaler, and an enema bag or container.

EXAMPLES

The examples described below are provided to illustrate the presentinvention and are not included for the purpose of limiting theinvention.

Example 1 Synthesis of [Ru(bpy)₂(4AP)₂]Cl₂

159 mg of Ru(bpy)₂Cl₂, where bpy=2,2′-bipyridine, were suspended in 7 mLof water at 85° C. under N₂. After dissolution, 66 mg of 4-aminopyridine(“4AP”) were added, and the resultant solution was heated for about 20minutes at about 50-80° C. or greater. A molar excess of NH₄PF₆, wasadded, and the resultant red solid was washed with water and dried. Thered solid was dissolved in a minimal amount of acetone, and to theacetone solution was added tetraethylammonium chloride, precipitating[Ru(bpy)₂(4AP)₂]Cl₂ (79% yield).

Example 2 Synthesis of [Ru(bpy)₂(TzGly)₂]Cl₂

[Ru(bpy)₂(TzGly)₂]Cl₂ was made according to the procedure used to make[Ru(bpy)₂(4AP)₂]Cl₂ set forth in Example 1, except that(RS)-(tetrazol-5-yl)glycine (“TzGly”) was used in place of 4AP.

Example 3 Synthesis of [Ru(bpy)₂(5HT)₂]Cl₂

[Ru(bpy)₂(5HT)₂]Cl₂ is made according to the procedure used to make[Ru(bpy)₂(4AP)₂]Cl₂ set forth in Example 1, except that serotonin(“5HT”) is used in place of 4AP.

Example 4 Synthesis of [Ru(bpy)₂(4AP)(PPh₃)]Cl₂

Ru(bpy)₂Cl₂, where bpy=2,2′-bipyridine, was suspended in water at aconcentration of 10 mg/mL at 85° C. under N₂. After dissolution, 1equivalent of PPh₃ was added, and the resultant solution was heated forabout 60 minutes at about 50-80° C. or greater. 1.1 Equivalents of 4APwere subsequently added, and heating continued for an additional 30minutes. A molar excess of NH₄PF₆, was added, and the resultant orangesolid was washed with water and dried. The orange was dissolved in aminimal amount of acetone, and to the acetone solution was addedtetraethylammonium chloride, precipitating [Ru(bpy)₂(4AP)(PPh₃)]Cl₂.

Example 5 Synthesis of [Ru(bpy)₂(TzGly)(PPh₃)]Cl₂

[Ru(bpy)₂(TzGly)(PPh₃)]Cl₂ was made according to the procedure used tomake [Ru(bpy)₂(4AP)(PPh₃)]Cl₂ set forth in Example 4, except that TzGlywas used in place of 4AP.

Example 6 Synthesis of [Ru(bpy)₂(5HT)(PPh₃)]Cl₂

[Ru(bpy)₂(5HT)(PPh₃)]Cl₂ is made according to the procedure used to make[Ru(bpy)₂(4AP)(PPh₃)]Cl₂ set forth in Example 4, except that serotoninis used in place of 4AP.

Example 7 Synthesis of [Ru(bpy)₂(nicotine)(PPh₃)]Cl₂

[Ru(bpy)₂(nicotine)(PPh₃)]Cl₂ is made according to the procedure used tomake [Ru(bpy)₂(4AP)(PPh₃)]Cl₂ set forth in Example 4, except thatnicotine is used in place of 4AP.

Example 8 Synthesis of [Ru(bpy)₂(TzGly)(py)]Cl₂

[Ru(bpy)₂(TzGly)(py)]Cl₂, where py=pyridine, was made according to theprocedure used to make [Ru(bpy)₂(4AP)(PPh₃)]Cl₂ set forth in Example 4,except that TzGly was used in place of 4AP and pyridine was used inplace of PPh₃.

Example 9 Synthesis of [Ru(bpy)₂(4AP)(py)]Cl₂

[Ru(bpy)₂(4AP)(py)]Cl₂, where py=pyridine, is made according to theprocedure used to make [Ru(bpy)₂(4AP)(PPh₃)]Cl₂ set forth in Example 4,except that pyridine is used in place of PPh₃.

Example 10 Synthesis of [Ru(bpy)₂(5HT)(py)]Cl₂

[Ru(bpy)₂(5HT)(py)]Cl₂, where py=pyridine, is made according to theprocedure used to make [Ru(bpy)₂(4AP)(PPh₃)]Cl₂ set forth in Example 4,except that 5HT is used in place of 4AP and pyridine is used in place ofPPh₃.

Example 11 Synthesis of [Ru(bpy)₂(nicotine)(py)]Cl₂

[Ru(bpy)₂(nicotine)(py)]Cl₂, where py=pyridine, is made according to theprocedure used to make [Ru(bpy)₂(4AP)(PPh₃)]Cl₂ set forth in Example 4,except that nicotine is used in place of 4AP and pyridine is used inplace of PPh₃.

Example 12 Synthesis of Co(DMG)₂(5HT)(Cl)

CoCl₂ was dissolved in a 1:1 v/v mixture of water/ethanol at a finalconcentration of about 0.2 M. Two equivalents of dimethylglyoxime(“DMG”) were added, and the resultant mixture was allowed to stir underN₂ until dissolution. One equivalent of 5HT was added, air was bubbledinto the resultant mixture for 6 hours and Co(DMG)₂(5HT)(Cl)precipitated. The precipitated product was filtered and washed.

Example 13 Photorelease of 4AP from [Ru(bpy)₂(4AP)₂]Cl₂

UV-vis spectra in water were obtained with an HP 8453 diode arrayspectrophotometer. RMN ¹H spectra were obtained using a Bruker 500 MHzequipment. CV measurements were performed with a PAR 273A potentiostat.Irradiation was effected by means of a pulsed Xe lamp, (pulse energy˜0.5 J), with a low-pass filter at 480 nm. Irradiation using a 473 nmDPSS laser gave similar results.

[Ru(bpy)₂(4AP)₂]Cl₂ is very soluble in water and stable in the dark,while undergoing decomposition under irradiation with visible light inits metal-to-ligand charge transfer (MLCT) band, centered at 489 nm. (InCH₃CN solution, the absorption band is red-shifted to 492 nm, consistentwith the lower polarity of the solvent, despite a previouscharacterization that reported 450 nm. However, light exposure of aCH₃CN solution of [Ru(bpy)₂(4AP)₂]Cl₂ produced a yellow compound withabsorption maximum at 450 nm. This may correspond to the previouslymisinterpreted assignments for this compound (D. Chun-Ying et al., 1999,J. Coord. Chem., 46:301-312), and the photoproduct is likely to be thecomplex [Ru(bpy)₂(4AP)CH₃CN]²⁺). Several ruthenium polypyridyl complexespresent this behavior. (D. V. Pinnick et al., 1984, Inorg. Chem.,23:1440-1445).

Although at pH 7 the spectrum of the irradiated complex is very similarto that of the original complex, a diminished shoulder at 470 nm becomesevident. To determine the nature of the photoreaction, NMR spectra weretaken before and after irradiation with visible light. FIG. 1 shows thesignal assigned to the meta hydrogens [Ru(bpy)₂(4AP)₂]Cl₂ (m1). Afterirradiation, this signal decreased, and two new signals appeared atlower fields: one corresponding to the free ligand (m3), and the othercorresponding to the aquo-4AP complex (m2), indicating photorelease ofthe 4AP. These two latter signals integrated for 0.30 and 0.27 of theinitial signal, which corresponds to a photoreaction of 60%.

The redox potential of the couple R^(III)/R^(II) for [Ru(bpy)₂(4AP)₂]Cl₂measured in water is E=0.76 V versus Ag/AgCl, which is consistent withthe higher basicity of 4AP compared with that of pyridine. Thus, theredox and the photochemistry of [Ru(bpy)₂(4AP)₂]Cl₂ is in totalagreement with results obtained corresponding to the Ru(bpy)₂XY family,X and Y being monodentate ligands. (See, e.g., E. S. Dodsworth et al.,1986, Chem. Phys. Lett., 124:152-158). The photoactivity of thesecompounds has been explained in terms of a reaction pathway thatinvolves the transition between the MLCT state to a lower-energy d-dstate, which promotes ligand release. There is a direct correspondencebetween the energy of the MLCT transition and the quantum yield of thephotoreaction. For [Ru(bpy)₂(4AP)CH₃CN]²⁺, the photoreaction yield isabout φ_(PR)=0.4. Since [Ru(bpy)₂(4AP)₂]Cl₂ presents a red-shifted band,a lower photoreaction yield is expected. An estimate based on earlyexperiments leads to an estimate of φ_(PR)≅0.02 at 473 nm.

Example 14 Neurophysiological Activity of 4AP Photoreleased from[Ru(bpy)₂(4AP)₂]Cl₂

A standard setup for intracellular voltage measurements was used, andthe medicinal leech Hirudo medicinalis was used to demonstratephotoreleased 4AP's neurophysiological activity. Hirudo medicinalis hasa central nerve cord with several ganglia, each one containing about 400neurons arranged in a known pattern. (W.-R. Schlue et al., 1980, J. Exp.Biol., 82:23-34). An entire ganglion was mounted on a dish. Thetransmembrane potential for a single cell (a neuron) in the ganglion wasrecorded by inserting inside the neuron a glass micropipet with amicrometer-sized end, filled with saturated aqueous KCl that acts as aluggin bridge for an Ag/AgCl electrode. Another Ag/AgCl electrode wasused as a reference. The signal was taken with an AM-System 1600amplifier, and the entire setup was covered with a Faraday cage. A 12bit A/D acquisition card was used to digitize the data using an ad-hocprogram written in QuickBasic.

Low Ca²⁺-high Mg²⁺ saline solution (NaCl, 102 mM; KCl, 4 mM; CaCl₂: 1mM; and MgCl₂: 10 mM; Tris base, pH 5.4 adjusted to 7.4) was perfusedthrough the dish. [Ru(bpy)₂(4AP)₂]Cl₂ and the free ligand 4AP wereinjected in the mainstream at controlled times. A pulsed Xe lamp locatedunder the dish was used to irradiate the solution. UV light was removedusing a band-pass filter at 500 nm. FIG. 2 shows the behavior of themembrane potential recorded at one of the Retzius (Rz) cells in theganglion. The upper graph in FIG. 2 shows the raw data, presentingperiods of rest potential and very fast spikes (action potentials),produced by the changes in membrane ion permeabilities. The lower graphshows the instantaneous spiking frequency at each time.

After impaling the cell with an electrode, many experiments wereperformed on the same cell to ensure reproducibility. After 5000seconds, the cell showed low activity, as can be seen at the left of thegraph. At t=5200 s, ˜100 μM Ru(bpy)₃Cl₂ was added to the salinesolution, without significant changes in activity. 300 seconds later, at5500 s, a light flash was directed to the ganglion. The sudden increasein the frequency of the action potentials is mainly due to thetemperature pulse, but after a short time the activity decreased to thebasal level. After washing by perfusion, further irradiation (t=6000 s)with a pulse showed a very similar pattern. At t=6250 s, ˜100 μM[Ru(bpy)₂(4AP)₂]Cl₂ was added to the saline and the activity remainedunchanged. However, after a new light flash (t=6400 s), sudden activitywas recorded and it remained high after 300 s. A second light pulse at6750 s promoted an even higher activity, which decreased only aftercleaning perfusion with pure saline.

A similar frequency increase occurred when free 4AP was perfused ontothe ganglion, thus demonstrating that the release of 4AP causes thismaintained frequency increase. Calibration of the cell activity usingsolutions of 4AP showed that in each irradiation, 10-15 μM of 4AP werereleased from [Ru(bpy)₂(4AP)₂]Cl₂ during the previous experiments.Neither toxicity nor a deleterious effect was observed on the neuronduring the experiments. These results show that a neuronal response canbe stimulated using [Ru(bpy)₂(4AP)₂]Cl₂, an illustrative PhotolabileCompound, to photorelease an organic molecule having neurophysiologicalactivity.

Example 15 Photorelease of TzGly from [Ru(bpy)₂(TzGly)(py)]Cl₂

The procedure for the photorelease of TzGly from[Ru(bpy)₂(TzGly)(py)]Cl₂ is analogous to that used for photorelease of4AP from [Ru(bpy)₂(4AP)₂]Cl₂ described above in Example 13, except thatthe irradiation light spot was very localized (diameter <1 micron).Irradiation of [Ru(bpy)₂(TzGly)(py)]Cl₂ at 470 nm photoreleased TzGly.

Example 16 Neurophysiological Activity of TzGly Photoreleased from[Ru(bpy)₂(TzGly)(py)]Cl₂

The neurophysiological activity of photoreleased TzGly was assessed byperforming experiments similar to those as set forth above in Example14. Accordingly, the standard setup for intracellular voltagemeasurements was used, and the medicinal leech Hirudo medicinalis wasused to demonstrate photoreleased TzGly's neurophysiological activity inthe leech ganglion.

Example 17 Synthesis of [Ru(bpy)₂(PMe₃)Cl)PF₆

520 mg of [Ru(bpy)₂Cl₂] were dissolved in 20 mL of a 1:1 mixture ofmethanol and water and refluxed under N₂. 1.2 mL of trimethylphosphine1M in THF (Aldrich, 324108) were added with a syringe. The reaction wasfollowed by UV-Vis spectroscopy. In some cases, more phosphine solutionwas added. Once the UV-Vis spectrum is stable, methanol and excessphosphine are distilled under vacuum with a rotavap. The resultingaqueous solution is filtered to remove any solids, and precipitated withexcess of KPF₆ over ice. The dark orange solid is washed three timeswith cold water and dried.

Example 18 Synthesis of [Ru(bpy)₂(PMe₃)GlutH₂](PF₆)₂

110 mg of [Ru(bpy)₂(PMe₃)Cl)PF₆ were dissolved in 2 mL of acetone. Asuspension of 200 mg chloride-containing anionic exchange resin DOWEX2×8 was added, and stirred during 10 minutes. The resulting[Ru(bpy)₂(PMe₃)Cl]Cl solution was filtered. 500 mg of monosodiumglutamate and 4.4 mL of NaOH 1M were added, and heated during 3 hours. 1mL of saturated KPF₆ was added, and the resulting precipitate wasdiscarded. The solution was cooled to 0° C. and acidified with HCl 5M topH2. [Ru(bpy)₂(PMe₃)GlutH₂](PF₆)₂ precipitates upon addition of excessof KPF₆. The yellowish-orange solid is washed three times with coldwater and dried.

Example 19 Photorelease of Glutamate from [Ru(bpy)₂(PMe₃)GlutH₂](PF₆)₂

[Ru(bpy)₂(PMe₃)GlutH₂](PF₆)₂ exhibits a bright orange color and presentsa high solubility in water at pH>6 (as the deprotonated species[Ru(bpy)₂(PMe3)Glu]). Its aqueous solutions present a strong metal toligand charge-transfer band (MLCT band) at 450 nm, characteristic ofthis family of Ru polypyridines. Cyclic voltammetry of the compounddissolved in acetonitrile (FIG. 15) shows three redox processes at 0.98,1.46 and 1.56 V vs. NHE, corresponding respectively to a Ru(III)/Ru(II)couple of the original complex, and those of the complexes bearingoxidation products of glutamate.

FIG. 16 (top) shows the UV-Vis spectrum of the complex, at pH=7 duringirradiation with a 450±20 nm LED. As the photoreaction proceeds, thegeneration of the aquo complex was recorded to reach completion in about4 minutes. The presence of two isosbestic points as well as a factoranalysis performed on the spectra indicate that just two colored speciesare present in the solution, according to what is expected in a singlephotoaquation process. The spectrum of the photoproduct was foundidentical to that of the complex [Ru(bpy)₂(PMe₃)(H₂O)]₂ ⁺, which can besynthesized by refluxing the chloro complex in water. At pH=11 a similarbehavior was observed, although in this case the product is the hydroxocomplex [Ru(bpy)₂(PMe₃)(OH)]⁺.

The inset in FIG. 16 (top) shows the yield of free glutamate generatedby the photolysis obtained from the analysis of the spectra. Theirradiance of the light source was calibrated using the known efficiencyof the photolysis of [Ru(bpy)(Py)₂]²⁺. The amount of photoreleasedglutamate was fitted using a two-parameter single-exponential function:y=a[1−exp(−bx)] and the quantum yield (φPC) was obtained as a•b,adopting a value of 0.13 at pH=7 and 0.10 at pH=11.

In order to estimate the uncaging time, a flash photolysis experimentwas performed on a basic aqueous solution of [Ru(bpy)₂(PMe₃)Glu] usingthe second harmonic of a Nd-YAGlaser (532 nm, 10 ns pulse). The resultsare shown in FIG. 16 (bottom). The photocleavage occurs within the first50 ns, being the fastest reported caged compound, to our knowledge. Thefact that a tens of nanoseconds are required to complete the glutamateuncaging reaction is compatible with the observation that the excitedstate lifetime measured for a similar compound containing pyridineinstead of glutamate is in the range of 10-100 ns.

The photoreaction was also followed by NMR spectroscopy. In the aromaticregion of the 1H-NMR spectrum of the complex (FIG. 17) it is possible todistinguish 16 signals corresponding to the bpy protons, which are foundduplicated because the obtained material is a roughly 1:1 mixture ofdiasteromeres, resulting from the coordination of L-glutamate with theracemic mixture of Λ and Δ Ru-bpy enantiomeres. No efforts were done toseparate the diasteromeres and both the chemical and biological testssuggest a similar behavior.

The aliphatic region corresponding to the glutamate moiety is depictedin the upper trace of FIG. 18, where —NH₂ protons appear at 4.50, 4.12,4.00 and 3.57 ppm. The presence of these signals in a D₂O solution,evidencing the absence of isotopic exchange in the complex, clearlyindicates that the coordination of glutamate is done via the aminenitrogen, hampering further protonation. This behavior differs from thatof free glutamate which, under the same conditions, exchanges rapidly D+for H+. After 2 months in D₂O at room temperature, no exchange is seen,also demonstrating that the compound is indeed inert.

After photolysis, many new signals appear in the aromatic region whichcorrespond to the aquo complexes. At the same time, the analysis of thealiphatic region (FIG. 18, bottom trace) clearly evidences theappearance of free glutamate signals. Coordinated —NH₂ signals diminishupon irradiation, and the characteristic signals of methylene protons offree glutamate appear at 3.50, 2.30 and 2.04 and 1.94 ppm.

Further addition of glutamate did not produce new signals, but only anintensity enhancement of the existent ones, which proves that the uniquealiphatic photoproduct is glutamate.

Biocompatibility tests were performed to evaluate the potential toxicityof the complex. 350 μM [Ru(bpy)₂(PMe₃)Glu], a concentration used fortwo-photon uncaging experiments, was incubated with living neocorticalpyramidal neurons in mouse brain slices, and carefully monitored themorphology and electrophysiological properties of the neurons usingtwo-photon microscopy and whole-cell patch recordings. After 1 hour ofincubation, no detectable effects were observed, and the neuronsremained healthy. This indicates that the complex does not modifymembrane integrity or has deleterious effect on living neurons.

All patent applications, published patent applications, issued andgranted patents, texts, and literature references cited in thisspecification are hereby incorporated herein by reference in theirentirety to more fully describe the state of the art to which thepresent invention pertains.

As various changes can be made in the above methods and compositionswithout departing from the scope and spirit of the invention asdescribed, it is intended that all subject matter contained in the abovedescription, shown in the accompanying drawings, or defined in theappended claims be interpreted as illustrative, and not in a limitingsense.

Example 20 A Ruthenium-Rhodamine Complex as an Activatable FluorescentProbe

This example describes a caged fluorescent probe based on aruthenium-bipyridine core, bearing a modified rhodamine as an activederivative of a labeling molecule. This complex behaves as a cagedfluorescent probe, increasing its fluorescence around 6-fold uponvisible light excitation. Thus, this example demonstrates the advantagesof Photolabile Compounds comprising a labeling molecule or an activederivative thereof in increasing fluorescence of fluorescent molecule oran active derivative thereof, such as, rhodamine B, rhodamine 6G,RhodB-MAPN, Rhod-6G-MAPN, RhodB-MAMePy and Rhod-6G-MAMePy.

All reagents were purchased from Sigma-Aldrich and used as received.Ru(bpy)₂Cl₂ was synthesized according to the literature using water assolvent (Viala, C.; Coudret, C. Inorg. Chim. Acta 2006, 359: 984-989).UV-vis spectra were taken with a HP8453 diode-array spectrometer. NMRspectra were obtained using a 500 MHz Bruker AM-500. Fluorescenceemission measurements were made with a PTI Quantamasterspectrofluorometer, corrected for the instruments response function.Rhodamine B was used as fluorescence standard of emission quantum yield(φ=0.31 in aqueous solutions). Quantum yield of the complex was obtaineddirectly from the ratio of the spectral areas, which present the sameshape.

The photouncaging quantum yield measurements were performed with aNd:YAG diode pumped solid state laser doubled to 473 nm with a constantpower of 6.3 mW. The light was collimated and sent through an opticalpath of 1 cm into a fluorescence glass cuvette, with stirring. Totalirradiation energy was measured using a Coherent Fieldmaster FM lightmeter with a visible light photodiode model SR45.

Microscopy of leech (Hirudo medicinalis) ganglia was performed in acustom-made setup for photoelectrophisiology equipped with amicromanipulator used to impale the neuron body with the ˜1 μm tipdiameter capillaries, and a CCD webcam as a registering device. Anisolated leech ganglion from a segment between 7 and 14 was pinned downin a sylgard-coated, 35 mm Petri dish. Retzius cells were identified bytheir position, size, and firing behavior. Intracellular recordings wereobtained with ˜20 MΩ sharp capillary microelectrodes, a Neuroprobe 1600(A-M Systems) amplifier and a A/D signal acquisition board at 1 kHzsampling rate and custom-made software. For iontophoretical injection ofthe dye the microelectrode was filled with recording solution to which 1mM caged fluorescent dye (chloride form) was added. Square, biphasicpulses of 1 nA, 2 Hz were applied for less that 5 min.

Images of thin spectrophotometric flow cells and FIA capillaries wereperformed using a Nikon TS-100 with a fluorescence adapter. Aconsumer-electronics type compact digital camera (Casio Exilim EX-FC100)focusing through a normal eyepiece through a custom adapter were use toregister the videos at ISO 800 sensitivity. Videos and image analysiswere done using public access ImageJ software. Activation offluorescence was performed with a 405 nm 6 mW laser diode was controlledwith a pulse generator using a fast Reed relay to provide 20 ms lightpulses. The laser beam was focused at the cell plane and monitored withan inverted microscope (Nikon TS100 w/fluorescence adapter).

Syntheses. Rhodamine B-Methylaminopropionitrileamide (RhodB-MAPN). 550mg of rhodamine B.HCl were dissolved into 10 mL of dry 1,2dichloroethane. The system was purged with N₂ and 300 μL of phosphorusoxychloride were added. The mixture was refluxed during 5 h. Thesolvents were distilled under a vacuum and the solid was immediatelydissolved in dry acetonitrile. 500 μL of triethylamine and 108 μL ofN-methylaminepropionitrile were added, and the mixture was refluxedduring 12 h. The solvent was removed under a vacuum, redissolved inwater, filtered to eliminate any solids and precipitated by addition ofexcess KPF₆. The dark red solid was washed several times with distilledwater and dried over silica gel. Exchange of Cl for PF₆ in order to havea water soluble ligand was performed by stirring overnight a 1:1acetone-water solution of RhodB-MAPN.PF₆ with Dowex 22 anionic resin andlyophilizing the obtained solution. RhodB-MAPN chloride salt is somewhathygroscopic and must be stored in a moisture free environment.

Overall yield: 55%. ¹H NMR (Acetone-d6): 1H δ 1.37 (t, 12H), 2.37 (t,2H), 3.16 (s, 3H), 3.53 (t, 2H), 3.80 (m, 8H), 6.98 (d, 2H), 7.20 (dd,2H), 7.37 (dd, 2H), 7.62 (m, 1H), 7.77 (m, 1H), 7.82 (t, 2H).

[Ru(bpy)₂(L)Cl]PF₆. For L=Rhodamine B-Methylaminopropionitrileamide

Twenty mg of Ru(bpy)₂Cl₂ were suspended in 2 mL of a 2:1 EtOH/watermixture and the suspension was heated to 80° C. until total dissolution.The formation of the [Ru(bpy)₂(H₂O)Cl]⁺ complex was determined by itsabsorption band at 490 nm (in water). After formation of the chloro-aquocomplex, two equivalents of the RhodB-MAPN chloride salt were added, andthe reaction was followed by TLC using silica plates and a mixture of1:1:1 water/EtOH/nBuOH as eluent. The solution was heated at 80° C.during about 2 h, until no further TLC changes were observed. All thefollowing procedures were done in darkness. The solution was filtered toremove any insoluble particles and the solvent was removed under avacuum. The obtained oil was redissolved in 4 mL of a 3:1acetone:methanol mixture and was precipitated by addition of excess THF.

The THF fraction containing unreacted ligand was discarded, and theprecipitate was washed several times with THF, redissolved in water andprecipitated with saturated KPF₆. Yield: 26%. NMR (Acetone-d6): 1H δ1.35 (m, 12H), 2.98 (t, 2H), 3.10 (s, 3H), 3.40-3.55 (dm, 2H), 3.80 (m,8H), 6.97 (d, 2H), 7.20-7.40 (m, 6H), 7.60 (d, 1H), 7.67 (t, 1H),7.70-7.85 (m, 4H), 7.88-7.98 (m, 4H), 8.17 (t, 1H), 8.30 (t, 1H), 8.56(dd, 2H), 8.69 (d, 1H), 8.72 (d, 1H), 9.55 (d, 1H), 10.08 (d, 1H).

For L=Vinylacetonitrile (VACN)

Twenty mg of Ru(bpy)₂Cl₂ were suspended in 2 mL of 96% EtOH, and thesuspension was heated at 80° C. until total dissolution. The formationof the [Ru(bpy)₂(H₂O)Cl]⁺ complex was determined by its band at 490 nmin water. 1.5 equivalents of VACN diluted in 4 mL of EtOH were added andthe mixture was kept at 80° C. during 2 h. The solvent was removed bydistilling under a vacuum, and the residue was redissolved in 2 mL ofwater, centrifuged to remove any solid, and precipitated with saturatedKPF₆. Yield 66%.

NMR (Acetone-d6): 1H δ 3.65 (d, 2H), 5.13 (d, 2H), 5.75 (m, 1H), 7.29(t, 1H), 7.32 (t, 1H), 7.70 (d, 1H), 7.81 (t, 1H), 7.92 (3t, 3H), 7.97(d, 1H), 8.20 (t, 1H), 8.31 (t, 1H), 8.56 (d, 1H), 8.60 (d, 1H), 8.69(d, 1H), 8.74 (d, 1H), 9.53 (d, 1H). 10.08 (d, 1H).

Rhodamines are useful fluorophores, due to their emission quantum yieldapproaching unity and their high resistance to photobleaching. They arecomprised of a xanthene moiety that provides the fluorescence and abenzoic acid which modulates their spectral properties. Scheme 1A showsthe structure of rhodamine B which was chosen as the starting point forthis example.

Ruthenium-bipyridyl complexes can easily be coordinated to donornitrogens such as those of the aliphatic amines, pyridines, imines, andeven nitriles, but not to those of anilines, not even primary ones.Diethylanilines of the rhodamine B do not form stable complexes with Rucenters and although carboxylates can be coordinated to Ru-bpy cores,such complexes are easily hydrolyzed in aqueous solutions at roomtemperature.

Given these properties, the chemical structure of rhodamine B wasmodified by adding a “sticky tail” in order to coordinate thefluorescent molecule to the metal center. In a first attempt tosynthesize a modified rhodamine bearing a coordinating group,carboxylate was amidated with aminopropionitrile, using the Adamczykprocedure (Adamczyk, M. J. Bioorg. Med. Chem. Lett. 2000, 10:1539-1541). The obtained compound is RhodB-APN, indicated in scheme 1B.However, although this ligand can be coordinated to Ru-bpy complexesthrough the nitrile, it undergoes isomerization to the cyclicspirolactame form at physiological pH (scheme 1C) becomingnonfluorescent. Rhodamine 6G can be modified similarly to the rhodamineB, described above.

A secondary amide cannot isomerize to the cyclic form, thus RhodB-MAPN,obtained through an amidation of rhodamine B acid chloride withaminopropionitrile (Scheme 1 D) presents a very high, yellowfluorescence while its terminal nitrile allows its coordination to aRu-bpy center.

The complex cis-[Ru(bpy)₂(L)Cl]PF₆, L) RhodB-MAPN was obtained as a darkpurple solid, slightly soluble in water and very soluble in acetone. Dueto its very high molar absorptivity, its water solubility is high enoughfor most experimental situations. For the FIA imaging experiments it wasused in this form. A much more soluble chloride salt, which is betterfor the biological experiments, was prepared by means of batch ionexchange with Dowex 22-Chloride resin in 1:1 acetone-water. The salt wasused in biological experiments. The solutions of the complex present aweak yellowish fluorescence. Preliminary studies showed that the complexis light sensitive, increasing its fluorescence when irradiated inaqueous, ethanol or acetone solutions.

FIG. 19 (top) shows the aliphatic region of the ¹H NMR spectrum of theRu complex bearing RhodB-MAPN as a ligand, showing the expected numberof signals and integrations for a coordinate RhodB-MAPN. The doublemultiplet at 3.40-3.55 ppm (labeled a′) corresponds to the methylenegroup closest to the coordination point. The triplet at 2.98 ppm (b′)and the singlet at 3.10 ppm (c′) correspond to the other methylene andthe N-methyl group respectively. Similar signals were obtained if justthe aminonitrile tail is coordinated to the Ru-bpy core. After 5 min ofirradiation inside the NMR tube using a 450 nm LED (middle trace), newsignals appear at 2.37, 3.16, and 3.53 ppm, due to the release of theRhodB-MAPN ligand. These three signals exactly match with those of thefree ligand (labeled a, c, and b, respectively, bottom trace). The fourethyl groups of RhodB-MAPN are not totally equivalent, and they are seenas a multiplet (—CH₂—) or broad triplet (—CH₃) and present little changeafter irradiation since they are far from the coordination center.

In the aromatic region the expected number of signals and integrationsfor a nonsymmetric cis complex bearing RhodB-MAPN were obtained. Afterphotolysis, the signals from the free RhodB-MAPN at 6.98, 7.20, 7.37,7.62, 7.77, and 7.82 appear at the same chemical shifts as those fromaquo-complex [Ru(bpy)₂(H₂O)Cl]⁺.

Dilute aqueous solutions of [Ru(bpy)₂(RhodB-MAPN)Cl]⁺ resent weakfluorescence with a maximum at 592 nm (excitation 518 nm).

FIG. 20 depicts the emission spectra of the complex during irradiation.The maximum and the shape of the initial emission spectrum is the sameas that of the free ligand RhodB-MAPN but the emission quantum yield ismuch lower, around pt=0.04. Quantum yields are easily calculatedintegrating the spectral areas. After irradiation with a 473 nm laser,RhodB-MAPN is released and the solution increases its fluorescence up to6 fold, to φt=0.24. The inset shows the maxima of the spectra as afunction of the irradiation time.

The quantum yield of rhodamine photorelease was calculated after theanalysis of fluorescence emission data during photouncaging at 473 nm. Acomplete spectrum was obtained once per second. Absorption of light bythe complex [Ru(bpy)₂(RhodB-MAPN)Cl]⁺ yields the aquo complex[Ru(bpy)₂(H₂O)Cl]⁺ and free RhodB-MAPN. In each irradiation period theamount of aquo complex and free RhodB-MAPN generated is given by thefollowing:δn=δtIφ _(PU)(1−10^(−Abs) T)Abs _(R) /Abs _(T)where δt is the irradiation period, I the light intensity in moles ofphotons per second, φ_(PU) is the quantum yield of photouncaging,Abs_(R) is the absorbance of the photoactive species[Ru(bpy)₂(RhodB-MAPN)Cl]⁺ at 473 nm and Abs_(T) is the total absorbanceof the irradiated solution. By means of finite element calculation ofthe former equation a photouncaging quantum yield of φ_(PU)=0.12 at 25°C. was calculated. This value is in good agreement with the reportedquantum yields for similar complexes.

The origin of this quenching can be attributed to the presence of thenearby ruthenium-bipyridine moiety, which presents an MLCT transitionwhich overlaps with that of the rhodamine emission. However, directinspection of the absorption bands of this complex at such a wavelengthrange is impossible, due to the very high molar extinction of thefluorescent ligand (ca., 105 M⁻¹ cm⁻¹), which obscures any absorptiondue to the metal center.

In order to measure this absorption, an analogous complex wassynthesized using vinylacetonitrile (VACN) instead of RhodB-MAPN. It isrecognized that the energy of the MLCT band of the Ru-bpy complexes arestrongly dependent on the nature and basicity of the ligands near the Rucenter but almost independent of the farther fragments. The complex[Ru(bpy)₂(VACN)Cl]⁺ shows a typical MLCT band, which is centered around460 nm and extends more than 100 nm to the low energy region,overlapping with the emission spectra of the rhodamine ligand. Thisabsorption is enough to explain the observed quenching. This analogcomplex photoreleases VACN with even a higher quantum yield ofφ_(PU)=0.21.

This example provides an activatable fluorescent probe with surprisingsensitivity in the visible light region. It increases its intrinsicfluorescence up to 6-fold after blue light irradiation. This probe isphysiologically friendly and can be injected into living cells—evenexcitable ones like neurons—with no sign of acute toxicity in short-term(˜2 h) experiments. The probe was used to image the laminar flow insidea thin spectrophotometric flow cell and to visualize theparabolic-shaped flow in a FIA capillary. This probe reveals not onlythe broadening of the plume, but also the memory effect due toaccumulation of analyte near the walls, where the flow velocityapproaches zero. In brief, the complex [Ru(bpy)₂(RhodB-MAPN)Cl]Cl is asurprisingly effective tool to image any kind of systems wheremanipulation of fluorescence is required.

[Ru(bpy)₂(Rhod6G-MAPN)Cl]Cl and [Ru(bpy)₂(RhodB-MAMePy)Cl] were madeusing the procedures described herein and exhibited results similar tothose described herein for [Ru(bpy)₂(RhodB-MAPN)Cl]Cl, thus illustratingthe applicability of the light antenna concept to the genus ofPhotolabile Compounds of Formulas I, II, III, V, VI, VII and VIIIdescribed herein, wherein L² comprises a labeling molecule or an activederivative thereof.

The same surprising properties demonstrated in this example for[Ru(bpy)₂(RhodB-MAPN)Cl]Cl are expected to apply to the PhotolabileCompounds of Formulas I, II, III, V, VI, VII and VIII described herein,wherein L² comprises a labeling molecule or an active derivativethereof.

Example 21 Synthesis of [Ru(bpy)₂(PMe₃)(MTG)]⁺²

A total of 67 mg of [Ru(bpy)₂(PMe₃)Cl]PF₆ were suspended in 4 ml acetoneand 2 ml water, and were heated and stirred at 80° C. until dissolution.Evaporated acetone was gradually replaced with water or acetone to keeptotal volume constant but avoiding precipitation. All the following wasperformed under dim red light. 100 mg of D(+)methylthiogalactose wereadded and the mixture was kept at 80° C. for 5 hours: The reactionprogress was followed by UV-Vis spectrophotometry until the aquo complexwas no longer detectable. The solution was cooled in ice-cold water andsaturated [BPh₄]⁻ as sodium salt was added carefully. The pale yellowprecipitate was washed six times in ice cold water by resuspending theprecipitate and centrifugation. NMR spectra were taken in a 500 MHzBruker Avance II spectrometer. Photolysis of the NMR sample wasperformed inside the unopened NMR tube, by means of a custom-built NMRphotolysis device based in 10 high current (100 mA) LEDs with emissionwavelength centered at 520+/−20 nm.

UV-Vis Absorption Spectrum of the Complex

[Ru(bpy)₂(PMe₃)(MTG)]⁺² is shown in FIG. 21 as the red line. The complexhas a molar absorptivity coefficient of ˜3100 and a maximum absorptionwavelength in the visible range at 422 nm. Photolysis quantum yield is0.3. Successive photolysis products of the same sample are shown inblack (FIG. 21).

Schematic drawing of [(Ru(bpy)₂P(Me)₃(MTG)]⁺² is shown in FIG. 22.Protons in caged MTG numbered in red correspond to signals labeled inthe NMR spectrum (FIG. 23). The first (top) tracing shows the aliphaticsignals of caged MTG. Middle trace corresponds to the photolysisproduct. Bottom trace shows free MTG. Signals below 2.5 ppm are shownmultiplied by (¼). The “signature” CH₃ group labeled (1) in MTG, usuallyat 2.25 ppm in free MTG, appears as a pair of signals (Δ & Λ isomers) at1.55 ppm and 1.48 ppm in the caged form. Also the proton labeled (2)appears as a pair of doublets at 4.07 ppm and 4.09 ppm in the complex,while in free MTG it appears as a doublet at 4.20 ppm. Solvent signalshave been truncated for clarity.

A handheld projector 3M (110 MPro) is used to irradiate for 20 minutesan E. coli culture growing in a piece of 7 cm diameter whatman filterpaper soaked in LB medium containing 1 mM [(Ru(bpy)₂P(Me)₃(MTG)]⁺² and0.02% X-Gal (FIG. 24A). After image projection, the culture is left at37° C. for until blue precipitate is noticed (˜40 minutes, FIG. 24B).

Example 22 Syntheses of Complexes of General Formula [Ru(Bpy)₂ L¹L²]⁺²

For L²=4-methylpyridine; L¹=benzonitrile:

67 mg (130 μmoles) Ru(bpy)₂Cl₂ were suspended in 10 ml ethanol and 2 mlwater, and were heated and stirred until dissolution. Under dim redlight 20 μl of 4-methylpyridine (220 μmoles) were added and the mixturewas refluxed for 3 hours. Volume was reduced in a rotary evaporator andthe reaction product was cooled and precipitated by addition of 500 μlof 0.5 M KPF₆ solution in water. The precipitate was washed in coldwater and dried in the dissicator overnight. This precipitate wasdissolved in 5 ml of acetone and 10 ml of water, and 5 equivalentsbenzonitrile were added. The product was hot-precipitated by adding 1 ml0.5 M of KPF₆ in water. The precipitate was filtered and washed withwater and ether. ¹H NMR (acetone-D₆) δ=9.91 (1H, d, (bpy)), 8.91 (1H, d,(bpy)), 8.88 (1H, d, (bpy)), 8.76 (1H, d, (bpy)), 8.75 (1H, d, (bpy)),8.68 (1H, d, (bpy)), 8.62 (1H), 8.44 (1H, t, (bpy)), 8.36 (1H, t,(bpy)), 8.14-8.2 (3H, m), 8.09 (1H, d, (bpy)), 8.02 (1H, t, (bpy)), 7.94(1H, t, (bpy)), 7.55-7.78 (3H, m), 7.55-7.61 (3H, m), 7.49 (1H, t,(bpy)), 7.37 (1H, d), 2.40 (3H, s, Me). The UV-vis spectrum of the[Ru(bpy)₂(4-methylpyridine)(benzonitrile)]²⁺ complex is shown in FIG.25. The spectrum corresponding to zero photolysis is the one with themaximum absorption peak furthest to the left. In this case, photolysisproducts have their maxima to the right of the original compound.

For L²=PMe₃; L¹=3-butenenitrile:

Starting from [Ru(bpy)₂(PMe₃)Cl]⁺ 3 equivalents of 3-butenenitrile wereadded; the remaining of the reaction proceeded as forL²=4-methylpyridine; L¹=benzonitrile. ¹H NMR (acetone-D₆) δ=9.50 (1H, d,(bpy)), 9.40 (1H, d, (bpy)), 8.86 (1H, d, (bpy)), 8.83 (1H, d, (bpy)),8.77 (1H, d, (bpy)), 8.68 (1H, d, (bpy)), 8.46-8.41 (2H, m), 8.23 (1H,t, (bpy)), 8.14 (1H, t, (bpy)), 8.07 (1H, d, (bpy)), 8.01 (1H, t,(bpy)), 7.97 (1H, t, (bpy)), 7.75 (1H, d, (bpy)), 7.58 (1H, t, (bpy)),7.48 (1H, t, (bpy)), 5.78-5.71 (1H, m, H₂C═CH—), 5.14 (1H, d, J=10 Hz,HH _(cis)C═CH—), 4.94 (1H, d, J=17 Hz, HH _(trans)C═CH—), 3.66-3.79 (2H,m, —CH ₂—CN), 1.32 (9H, d, J_(HP)=9 Hz, PMe₃). The UV-vis spectrum ofthe [Ru(bpy)₂(PMe₃)(3-butenenitrile)]²⁺ complex is shown in FIG. 26. Thespectrum corresponding to zero photolysis is the one with the maximumabsorption peak furthest to the left. In this case, photolysis productshave their maxima to the right of the original compound.

For L²=PPh₃; L¹=butenenitrile:

Starting from [Ru(bpy)₂(PPh₃)Cl]⁺ 3 equivalents of 3-butenenitrile wereadded; the remaining of the reaction proceeded as forL²=4-methylpyridine; L¹=benzonitrile. ¹H NMR (acetone-D₆) δ=9.24 (1H, d,(bpy)), 8.92 (1H, d, (bpy)), 8.89 (1H, d, (bpy)), 8.82 (1H, d, (bpy)),8.65 (1H, d, (bpy)), 8.63 (1H, d, (bpy)), 8.43 (1H, t, (bpy)), 8.27 (1H,t, (bpy)), 8.20 (1H, t, (bpy)), 8.10 (1H, t, (bpy)), 7.79 (1H, t,(bpy)), 7.55-7.63 (3H, m), 7.47-7.53 (4H, m), 7.29-7.39 (12H, m), 7.26(1H, t, (bpy)), 5.63-5.73 (1H, m, H₂C═CH—), 5.16 (1H, d, J=10.5 Hz, HH_(cis)C═CH—), 4.97 (1H, d, J=17 Hz, HH _(trans)C═CH—), 3.71-3.75 (2H, m,—CH ₂—CN). The UV-vis spectrum of the[Ru(bpy)₂(PPh₃)(3-butenenitrile)]²⁺ complex is shown in FIG. 27. Thespectrum corresponding to zero photolysis is the one with the maximumabsorption peak furthest to the left. In this case, photolysis productshave their maxima to the right of the original compound.

For L²=PMe₃; L¹=2-cyanophenol:

Starting from [Ru(bpy)₂(PPh₃)Cl]⁺ 3 equivalents of 2-cyanophenol wereadded; the remaining of the reaction proceeded as forL²=4-methylpyridine; L¹=benzonitrile. ¹H NMR (acetone-D₆) δ=9.73 (1H, d,(bpy)), 9.45 (1H, d, (bpy)), 8.85 (2H, d, (bpy)), 8.76 (1H, d, (bpy)),8.70 (1H, d, (bpy)), 8.41-8.44 (2H, m, (bpy)), 8.21 (1H, t, (bpy)), 8.16(1H, t, (bpy)), 8.12 (1H, d, (bpy)), 8.00 (1H, t, (bpy)), 7.96 (1H, t,(bpy)), 7.78 (1H, d, (bpy)), 7.58 (1H, t), 7.47-7.54 (1H, m), 7.44 (1H,t), 7.38 (1H, d), 6.97 (1H, d), 6.77 (1H, t), 1.37 (9H, d, J_(HP)=9 Hz,PMe₃). The UV-vis spectrum of the [Ru(bpy)₂(PMe₃)(2-cyanophenol)]²⁺complex is shown in FIG. 28. The spectrum corresponding to zerophotolysis is the one with the maximum absorption peak furthest to theleft. In this case, photolysis products have their maxima to the rightof the original compound.

Analogously to the above procedures, the following complexes were alsosynthesized:

The UV-vis spectrum of the [Ru(bpy)₂(4-methylpyridine)(2-cyanophenol)]²⁺complex is shown in FIG. 29. The spectrum corresponding to zerophotolysis is the one with the maximum absorption peak furthest to theleft. In this case, photolysis products have their maxima to the rightof the original compound.

The UV-vis spectrum of the[Ru(bpy)₂(4-methylpyridine)(3-butenenitrile)]²⁺ complex is shown in FIG.30. The spectrum corresponding to zero photolysis is the one with themaximum absorption peak furthest to the left. In this case, photolysisproducts have their maxima to the right of the original compound.

The UV-vis spectrum of the [Ru(bpy)₂(PPh₃)(2-cyanophenol)]²⁺ complex isshown in FIG. 31. The spectrum corresponding to zero photolysis is theone with the maximum absorption peak furthest to the left. In this case,photolysis products have their maxima to the right of the originalcompound.

It will be noted that, photolysis products to the right of the reactivesis not a general rule. In some cases, photolysis absorption spectra maylie to the left of the reactant spectrum.

We claim:
 1. A compound of Formula V:

wherein: each L¹ is independently an organic molecule having: (a) a5-membered monocyclic aromatic ring, one of the ring's members being anitrogen atom that forms a bond with Ru; (b) a 6-membered monocyclicaromatic ring, one of the ring's members being a nitrogen atom thatforms a bond with Ru; (c) an 8-10-membered bicyclic ring, one of thebicyclic rings being aromatic and having a nitrogen atom member thatforms a bond with Ru; (d) an —NH₂ group whose nitrogen atom forms a bondwith Ru; (e) a —COOH group, one of whose oxygen atoms forms a bond withRu; (f) a —PR₂ group whose phosphorus atom forms a bond with Ru, whereinR is independently —H, —C₁-C₁₈ alkyl, or aryl; (g) an —SR group whosesulfur atom forms a bond with Ru, wherein R is independently —H, —C₁-C₁₈alkyl, or aryl; or (h) a —CN group whose nitrogen atom forms a bond withRu; L² is (R⁹)₃P, or (R⁹O)₃P and m is 2; or L² is a labeling molecule oran active derivative thereof connected to Ru through the phosphorousatom of (R⁹)₂P or (R⁹O)₂P and m is 2; or L² is a labeling molecule or anactive derivative thereof connected to Ru through the nitrogen atom of:NHR⁹, N(R⁹)₂, pyridyl, C(R⁹)═NH, C(R⁹)═NR⁹, cyclic aliphatic amine groupor nitrile and m is 2; wherein each R⁹ is independently —C₁-C₁₈ alkyl,—C₃-C₈ cycloalkyl, or phenyl; wherein when L² is P(phenyl)₃ or alabeling molecule or an active derivative thereof connected to Ruthrough the phosphorous atom of P(phenyl)₂, each phenyl is independentlysubstituted with —C₁-C₁₈ alkyl, —(C₁-C₁₈ alkyl)-OH, aryl, —(C₁-C₁₈alkyl)-oxy, —(C₁-C₁₈ alkyl)-amino, —(C₁-C₁₈ alkyl)-thio, —CO₂Y, —C(═O)Y,—C(═O)NY₂, —NH₂, —NO₂, —OH, or —SH, and R¹ to R⁴ are independently —H,—C₁-C₁₈ alkyl; —NH₂, —(C₁-C₁₈ alkyl)-O—(C₁-C₁₈ alkyl), —OC(O)(C₁-C₁₈alkyl), —(C₁-C₁₈ alkyl)-OH, aryl, —(C₁-C₁₈ alkyl)-oxy, —(C₁-C₁₈alkyl)-amino, —(C₁-C₁₈ alkyl)-thio, —CO₂Y, —C(═O)Y, —C(═O)NY₂, —NO₂, or—SH, or R¹ and R² and/or R³ and R⁴ can combine to form a carbocyclicring substituted by one or more oxo groups; wherein when L² is notP(phenyl)₃, R¹ to R⁴ are independently —H, —(C₁-C₁₈ alkyl)-OH, aryl,—(C₁-C₁₈ alkyl)-oxy, —(C₁-C₁₈ alkyl)-amino, —(C₁-C₁₈ alkyl)-thio, —CO₂Y,—C(═O)Y, —C(═O)NY₂, —NO₂, —OH, or —SH, or R¹ and R² and/or R³ and R⁴ cancombine to form a carbocyclic ring substituted by one or more oxogroups, wherein at least one of R¹ to R⁴ is not H; X is Cl⁻, F⁻, Br⁻,I⁻, PF₆ ⁻, CF₃SO₃ ⁻, (C₁-C₁₈ alkyl)-CO₂ ⁻, or (C₁-C₁₈ alkyl)-SO₃ ⁻; andY is selected from the group consisting of —H, —C₁-C₁₈ alkyl, aryl,—(C₁-C₁₈ alkyl)-aryl, —C₃-C₈ cycloalkyl, heteroaryl, and heterocyclyl.2. The compound of claim 1, wherein the organic molecule is4-aminopyridine.
 3. The compound of claim 1, wherein the organicmolecule is (RS)-(tetrazol-5-yl) glycine.
 4. The compound of claim 1,wherein the organic molecule is (tetrazol-5-yl) AMPA.
 5. The compound ofclaim 1, wherein the organic molecule is nicotine or caffeine.
 6. Thecompound of claim 1, wherein the organic molecule is serotonin,epinephrine, norepinephrine, or dopamine.
 7. The compound of claim 1,wherein the organic molecule is adenosine 5′-diphosphate ADP, adenosine5′-triphosphate ATP, adenosine 5′-monophosphate AMP, cyclic adenosine5′-diphosphate ribose, or adenosine 3′, 5′-cyclic monophosphate.
 8. Thecompound of claim 1, wherein the organic molecule is aminobutyric acidor L-glutamic acid, or methyl-D-aspartic acid.
 9. The compound of claim1, wherein the organic molecule is methylbeta-D-1-thiogalactopyranoside.
 10. The compound of claim 1, wherein theorganic molecule is isopropyl beta-D-1-thiogalactopyranoside.
 11. Thecompound of claim 1, wherein the organic molecule is mercaptopurine,thioguanine, doxorubicin, cytarabin, temozolomide or gentamicin.
 12. Thecompound of claim 1, wherein the organic molecule is mercaptopurine. 13.The compound of claim 1, wherein the organic molecule is thioguanine.14. The compound of claim 1, wherein the organic molecule isdoxorubicin.
 15. The compound of claim 1, wherein the organic moleculeis cytarabin.
 16. The compound of claim 1, wherein the organic moleculeis temozolomide.
 17. The compound of claim 1, wherein the organicmolecule is gentamicin.
 18. The compound of claim 1, wherein the organicmolecule is benzonitrile, 3-butenenitrile or 2-cyanophenol.
 19. A methodfor releasing an organic molecule from a Photolabile Compound,comprising: exposing a compound of claim 1 to light under conditionssufficient to release the organic molecule.
 20. The method of claim 19,wherein the light comprises a wavelength of about 300 to about 500 nm.21. The method of claim 19, wherein the light comprises a wavelength ofabout 300 to about 360 nm.
 22. The method of claim 19, wherein the lightcomprises a wavelength of about 450 to about 500 nm.
 23. The method ofclaim 19, wherein the light comprises visible light or infrared light.24. The method of claim 19, wherein the exposing occurs at a temperaturefrom about 0° C. to about 150° C.
 25. A method for making an organicmolecule bioavailable to a subject, comprising: (a) administering acompound of claim 1 to the subject; and (b) exposing the compound tolight under conditions sufficient to release the organic molecule fromthe compound, wherein the organic molecule has: a 5-membered monocyclicaromatic ring, one of the ring's members being a nitrogen atom thatforms a bond with Ru; (ii) a 6-membered monocyclic aromatic ring, one ofthe ring's members being a nitrogen atom that forms a bond with Ru;(iii) an 8-10-membered bicyclic ring, one of the bicyclic rings beingaromatic and having a nitrogen atom member that forms a bond with Ru;(iv) an —NH₂ group whose nitrogen atom forms a bond with Ru; (v) a —COOHgroup, one of whose oxygen atoms forms a bond with Ru; (vi) a —PR₂ groupwhose phosphorus atom forms a bond with Ru, wherein R is independently—H, —C₁-C₁₈ alkyl, or aryl; (vii) an —SR group whose sulfur atom forms abond with Ru, wherein R is independently —H, —C₁-C₁₈ alkyl, or aryl; or(viii) a —CN group whose nitrogen atom forms a bond with Ru.
 26. Themethod of claim 25, wherein the organic molecule has: a 5-memberedmonocyclic aromatic ring, one of the ring's members being a nitrogenatom that forms a bond with Ru; (ii) a 6-membered monocyclic aromaticring, one of the ring's members being a nitrogen atom that forms a bondwith Ru; (iii) an 8-10-membered bicyclic ring, one of the bicyclic ringsbeing aromatic and having a nitrogen atom member that forms a bond withRu; (iv) an —NH₂ group whose nitrogen atom forms a bond with Ru; (v) a—COOH group, one of whose oxygen atoms forms a bond with Ru; (vi) a —PR₂group whose phosphorus atom forms a bond with Ru, wherein R isindependently —H, —C₁-C₁₈ alkyl, or aryl; or (vii) an —SR group whosesulfur atom forms a bond with Ru, wherein R is independently —H, —C₁-C₁₈alkyl, or aryl.
 27. The method of claim 25, wherein the light issunlight, photo-optic light, or laser light.
 28. The method of claim 25,wherein the light is visible light or infrared light.
 29. The method ofclaim 25, wherein the exposing occurs at the site of a tumor, cancer, orneoplasm.
 30. The method of claim 25, wherein the exposing occurs at thesite of a blood dyscrasia.
 31. The method of claim 25, wherein theadministering occurs intravenously, topically, intradermally,intramuscularly, transdermally, subcutaneously, intranasally,parenterally, intrathecally, vaginally, rectally, colorectally, orally,intracranially, retroorbitally, intrasternally, or by injection.
 32. Themethod of claim 25, wherein the administering is via a transdermalpatch.
 33. A composition comprising a compound of claim 1 and aphysiologically acceptable carrier, vehicle, diluent, or excipient. 34.A vessel containing a compound of claim
 1. 35. The vessel of claim 34,further containing a biological sample.
 36. The vessel of claim 35,wherein the biological sample is an organ, tissue, cell, or hair sample.37. The vessel of claim 36, wherein the tissue is neuronal tissue. 38.The vessel of claim 36, wherein the cell is a neuronal cell.
 39. Thevessel of claim 36, wherein the tissue or cell is a tumor, cancer, orneoplastic tissue or cell.
 40. The vessel of claim 35, wherein thebiological sample is a body fluid sample.
 41. The vessel of claim 40,wherein the body fluid sample is blood, serum, plasma, lymph, saliva,sputum, tears, semen, or urine.
 42. A kit comprising a compound of claim1 and instructions for use of the compound.
 43. A method for enhancingthe solubility of an organic molecule, comprising complexing an organicmolecule to a photolabile caging group to form a compound of claim 1;wherein exposing the compound to light under sufficient conditionsreleases the organic molecule from the compound, and wherein the organicmolecule has: (i) a 5-membered monocyclic aromatic ring, one of thering's members being a nitrogen atom that forms a bond with Ru; (ii) a6-membered monocyclic aromatic ring, one of the ring's members being anitrogen atom that forms a bond with Ru; (iii) an 8-10-membered bicyclicring, one of the bicyclic rings being aromatic and having a nitrogenatom member that forms a bond with Ru; (iv) an —NH₂ group whose nitrogenatom forms a bond with Ru; (v) a —COOH group, one of whose oxygen atomsforms a bond with Ru; (vi) a —PR₂ group whose phosphorus atom forms abond with Ru, wherein R is independently —H, —C₁-C₁₈ alkyl, or aryl;(vii) an —SR group whose sulfur atom forms a bond with Ru, wherein R isindependently —H, —C₁-C₁₈ alkyl, or aryl; or (viii) a —CN group whosenitrogen atom forms a bond with Ru.
 44. The method of claim 43, whereinthe organic molecule has: (i) a 5-membered monocyclic aromatic ring, oneof the ring's members being a nitrogen atom that forms a bond with Ru;(ii) a 6-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with Ru; (iii) an 8-10-memberedbicyclic ring, one of the bicyclic rings being aromatic and having anitrogen atom member that forms a bond with Ru; (iv) an —NH₂ group whosenitrogen atom forms a bond with Ru; (v) a —COOH group, one of whoseoxygen atoms forms a bond with Ru; (vi) a —PR₂ group whose phosphorusatom forms a bond with Ru, wherein R is independently —H, —C₁-C₁₈ alkyl,or aryl; or (vii) an —SR group whose sulfur atom forms a bond with Ru,wherein R is independently —H, —C₁-C₁₈ alkyl, or aryl.
 45. A compound ofFormula VI:

wherein: each L¹ is independently an organic molecule having: (a) a5-membered monocyclic aromatic ring, one of the ring's members being anitrogen atom that forms a bond with Ru; (b) a 6-membered monocyclicaromatic ring, one of the ring's members being a nitrogen atom thatforms a bond with Ru; (c) an 8-10-membered bicyclic ring, one of thebicyclic rings being aromatic and having a nitrogen atom member thatforms a bond with Ru; (d) an —NH₂ group whose nitrogen atom forms a bondwith Ru; (e) a —COOH group, one of whose oxygen atoms forms a bond withRu; (f) a —PR₂ group whose phosphorus atom forms a bond with Ru, whereinR is independently —H, —C₁-C₁₈ alkyl, or aryl; (g) an —SR group whosesulfur atom forms a bond with Ru, wherein R is independently —H, —C₁-C₁₈alkyl, or aryl; or (h) a —CN group whose nitrogen atom forms a bond withRu; L² is P(phenyl)₃, wherein each phenyl is independently substitutedwith —C₁-C₁₈ alkyl, —(C₁-C₁₈ alkyl)-OH, aryl, —(C₁-C₁₈ alkyl)-oxy,—(C₁-C₁₈ alkyl)-amino, —(C₁-C₁₈ alkyl)-thio, —CO₂Y, —C(═O)Y, —C(═O)NY₂,—NH₂, —NO₂, —OH, or —SH; or L² is a labeling molecule or an activederivative thereof connected to Ru through the phosphorous atom of(R⁹)₂P or (R⁹O)₂P and m is 2; or L² is a labeling molecule or an activederivative thereof connected to Ru through the nitrogen atom of: NHR⁹,N(R⁹)₂, pyridyl, C(R⁹)═NH, C(R⁹)═NR⁹, cyclic aliphatic amine group ornitrile and m is 2; wherein each R⁹ is independently —C₁-C₁₈ alkyl,—C₃-C₈ cycloalkyl, or phenyl; R¹ to R⁴ are independently —H, —C₁-C₁₈alkyl; —NH₂, —(C₁-C₁₈ alkyl)-O—(C₁-C₁₈ alkyl), —OC(O)(C₁-C₁₈ alkyl),—(C₁-C₁₈ alkyl)-OH, aryl, —(C₁-C₁₈ alkyl)-oxy, —(C₁-C₁₈ alkyl)-amino,—(C₁-C₁₈ alkyl)-thio, —CO₂Y, —C(═O)Y, —C(═O)NY₂, —NO₂, or —SH, or R¹ andR² and/or R³ and R⁴ can combine to form a carbocyclic ring substitutedby one or more oxo groups; X is Cl⁻, F⁻, Br⁻, I⁻, PF₆ ⁻, CF₃SO₃ ⁻,(C₁-C₁₈ alkyl)-CO₂ ⁻, or (C₁-C₁₈ alkyl)-SO₃ ⁻; and Y is selected fromthe group consisting of —H, —C₁-C₁₈ alkyl, aryl, —(C₁-C₁₈ alkyl)-aryl,—C₃-C₈ cycloalkyl, heteroaryl, and heterocyclyl.
 46. The compound ofclaim 45, wherein each L¹ is independently an organic molecule having:(a) a 5-membered monocyclic aromatic ring, one of the ring's membersbeing a nitrogen atom that forms a bond with Ru; (b) a 6-memberedmonocyclic aromatic ring, one of the ring's members being a nitrogenatom that forms a bond with Ru; (c) an 8-10-membered bicyclic ring, oneof the bicyclic rings being aromatic and having a nitrogen atom memberthat forms a bond with Ru; (d) an —NH₂ group whose nitrogen atom forms abond with Ru; (e) a —COOH group, one of whose oxygen atoms forms a bondwith Ru; (f) a —PR₂ group whose phosphorus atom forms a bond with Ru,wherein R is independently —H, —C₁-C₁₈ alkyl, or aryl; or (g) an —SRgroup whose sulfur atom forms a bond with Ru, wherein R is independently—H, —C₁-C₁₈ alkyl, or aryl.
 47. The compound of claim 45, wherein L² isP(phenyl)₃ and each phenyl is independently substituted at the 3 or 4position.
 48. The compound of claim 45, wherein L² is P(phenyl)₃ and atleast one phenyl is substituted with —(C₁-C₁₈ alkyl)-OH.
 49. Thecompound of claim 45, wherein L² is P(phenyl)₃ and each phenyl issubstituted with —(C₁-C₁₈ alkyl)-OH.
 50. The compound of claim 45,wherein L² is P(phenyl)₃ and at least one phenyl is substituted with—COOH.
 51. The compound of claim 45, wherein L² is P(phenyl)₃ and eachphenyl is substituted with —COOH.
 52. The compound of claim 45, whereinL² is P(phenyl)₃ and at least one phenyl is substituted with —OH. 53.The compound of claim 45, wherein L² is P(phenyl)₃ and each phenyl issubstituted with —OH.
 54. The compound of claim 45, wherein L² isP(phenyl)₃ and at least one phenyl is substituted with —NH₂.
 55. Thecompound of claim 45, wherein L² is P(phenyl)₃ and each phenyl issubstituted with —NH₂.
 56. A compound of Formula VII:

wherein: each L¹ is independently an organic molecule having: (a) a5-membered monocyclic aromatic ring, one of the ring's members being anitrogen atom that forms a bond with Ru; (b) a 6-membered monocyclicaromatic ring, one of the ring's members being a nitrogen atom thatforms a bond with Ru; (c) an 8-10-membered bicyclic ring, one of thebicyclic rings being aromatic and having a nitrogen atom member thatforms a bond with Ru; (d) an —NH₂ group whose nitrogen atom forms a bondwith Ru; (e) a —COOH group, one of whose oxygen atoms forms a bond withRu; (f) a —PR₂ group whose phosphorus atom forms a bond with Ru, whereinR is independently —H, —C₁-C₁₈ alkyl, or aryl; (g) an —SR group whosesulfur atom forms a bond with Ru, wherein R is independently —H, —C₁-C₁₈alkyl, or aryl; or (h) a —CN group whose nitrogen atom forms a bond withRu; L² is (R⁹)₃P, (R⁹O)₃P, m is 2, and L² is not P(phenyl)₃; or L² is alabeling molecule or an active derivative thereof connected to Ruthrough the phosphorous atom of (R⁹)₂P or (R⁹O)₂P and m is 2; or L² is alabeling molecule or an active derivative thereof connected to Ruthrough the nitrogen atom of: NHR⁹, N(R⁹)₂, pyridyl, C(R⁹)═NH,C(R⁹)═NR⁹, cyclic aliphatic amine group or nitrile and m is 2; whereineach R⁹ is independently —C₁-C₁₈ alkyl, —C₃-C₈ cycloalkyl, or phenyl; R¹to R⁴ are independently —H, —(C₁-C₁₈ alkyl)-OH, aryl, —(C₁-C₁₈alkyl)-oxy, —(C₁-C₁₈ alkyl)-amino, —(C₁-C₁₈ alkyl)-thio, —CO₂Y, —C(═O)Y,—C(═O)NY₂, —NO₂, —OH, or —SH, or R¹ and R² and/or R³ and R⁴ can combineto form a carbocyclic ring substituted by one or more oxo groups,wherein at least one of R¹ to R⁴ is not H; X is Cl⁻, F⁻, Br⁻, I⁻, PF₆ ⁻,CF₃SO₃ ⁻, (C₁-C₁₈ alkyl)-CO₂ ⁻, or (C₁-C₁₈ alkyl)-SO₃ ⁻; and Y isselected from the group consisting of —H, —C₁-C₁₈ alkyl, aryl, —(C₁-C₁₈alkyl)-aryl, —C₃-C₈ cycloalkyl, heteroaryl, and heterocyclyl.
 57. Thecompound of claim 56, wherein each L¹ is independently an organicmolecule having: (a) a 5-membered monocyclic aromatic ring, one of thering's members being a nitrogen atom that forms a bond with Ru; (b) a6-membered monocyclic aromatic ring, one of the ring's members being anitrogen atom that forms a bond with Ru; (c) an 8-10-membered bicyclicring, one of the bicyclic rings being aromatic and having a nitrogenatom member that forms a bond with Ru; (d) an —NH₂ group whose nitrogenatom forms a bond with Ru; (e) a —COOH group, one of whose oxygen atomsforms a bond with Ru; (f) a —PR₂ group whose phosphorus atom forms abond with Ru, wherein R is independently —H, —C₁-C₁₈ alkyl, or aryl; or(g) an —SR group whose sulfur atom forms a bond with Ru, wherein R isindependently —H, —C₁-C₁₈ alkyl, or aryl.
 58. The compound of claim 56,wherein L² is P(methyl)(phenyl)₂.
 59. The compound of claim 56, whereinL² is P(methyl)₂(phenyl).
 60. A compound of Formula VIII:

wherein: each L¹ is independently a labeling molecule or an activederivative thereof connected to Ru through the phosphorous atom of(R⁹)₂P or (R⁹O)₂P and m is 2; or L¹ is a labeling molecule or an activederivative thereof connected to Ru through the nitrogen atom of: NHR⁹,N(R⁹)₂, pyridyl, C(R⁹)═NH, C(R⁹)═NR⁹, cyclic aliphatic amine group ornitrile and m is 2; wherein each R⁹ is independently —C₁-C₁₈ alkyl,—C₃-C₈ cycloalkyl, or phenyl; L² is Cl—, phosphine, OH₂, or pyridine andm is 2; or L² is —CN and m is 1; R¹ to R⁴ are independently —H, —C₁-C₁₈alkyl; —NH₂, —(C₁-C₁₈ alkyl)-O—(C₁-C₁₈ alkyl), —OC(O)(C₁-C₁₈ alkyl),—(C₁-C₁₈ alkyl)-OH, aryl, —(C₁-C₁₈ alkyl)-oxy, —(C₁-C₁₈ alkyl)-amino,—(C₁-C₁₈ alkyl)-thio, —CO₂Y, —C(═O)Y, —C(═O)NY₂, —NO₂, or —SH, or R¹ andR² and/or R³ and R⁴ can combine to form a carbocyclic ring substitutedby one or more oxo groups; X is Cl⁻, F⁻, Br⁻, I⁻, PF₆ ⁻, CF₃SO₃ ⁻,(C₁-C₁₈ alkyl)-CO₂ ⁻, or (C₁-C₁₈ alkyl)-SO₃ ⁻; and Y is selected fromthe group consisting of —H, —C₁-C₁₈ alkyl, aryl, —(C₁-C₁₈ alkyl)-aryl,—C₃-C₈ cycloalkyl, heteroaryl, and heterocyclyl.
 61. The compound ofclaim 60, wherein the labeling molecule or an active derivative thereofcontains a —CN group whose nitrogen atom forms a bond with Ru.
 62. Thecompound of claim 60, wherein the labeling molecule or an activederivative thereof contains rhodamine.
 63. The compound of claim 60,wherein the labeling molecule or an active derivative thereof containsfluorescein.
 64. The compound of claim 60, wherein the labeling moleculeor an active derivative thereof contains iodeosin.
 65. A method forreleasing a fluorescent molecule from a Photolabile Compound,comprising: exposing a compound of claim 60 to light under conditionssufficient to release the fluorescent molecule.
 66. The method of claim65, wherein the light comprises a wavelength of about 300 to about 500nm.
 67. The method of claim 65, wherein the light comprises a wavelengthof about 300 to about 360 nm.
 68. The method of claim 65, wherein thelight comprises a wavelength of about 450 to about 500 nm.
 69. Themethod of claim 65, wherein the light comprises visible light orinfrared light.
 70. The method of claim 65, wherein the exposing occursat a temperature from about 0° C. to about 150° C.
 71. The method of anyone of claims 1, 45, 56 or 60 wherein L² is a labeling molecule or aderivative thereof.
 72. The method of claim 71, wherein L² is RhodamineB-Methylaminopropionitrileamide (RhodB-MAPN), Rhodamine 6G-Methylaminopropionitrileamide (Rhod6G-MAPN), RhodB-MAMePy, Rhod6G-MAMePy or asalt thereof.
 73. A compound selected from the group consisting of:[Ru(bpy)₂(RhodB-MAPN)Cl]Cl, [Ru(bpy)₂(Rhod6G-MAPN)Cl]Cl,[Ru(bpy)₂(RhodB-MAMePy)Cl]Cl.
 74. A compound selected from the groupconsisting of: [Ru(bpy)₂(RhodB-MAPN)Cl]Z, [Ru(bpy)₂(Rhod6G-MAPN)Cl]Z,[Ru(bpy)₂(RhodB-MAMePy)Cl]Z, wherein Z is an anion.
 75. The compound ofclaim 45, wherein L² is P(phenyl)₃ and at least one phenyl issubstituted with —NO₂.
 76. The compound of claim 45, wherein L² isP(phenyl)₃ and each phenyl is substituted with —NO₂.